Small cationic antimicrobial peptides

ABSTRACT

The present invention relates generally to peptides and more specifically to antimicrobial and immunomodulatory host defense peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/CA2007/001453, filed Aug. 21, 2007, and claims priority to U.S.Application No. 60/839,253, filed Aug. 21, 2006, which are incorporatedby reference herein in their entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 14386 Sequence Listing.txt, created on Jun. 7, 2011,with a size of 257 kb and comprising 1268 sequences. The sequencelisting is hereby incorporated by reference.

FIELD

The present invention relates generally to peptides and morespecifically to antimicrobial and immunomodulatory host defensepeptides.

BACKGROUND

The treatment of bacterial infections with antibiotics is one of themainstays of human medicine. Unfortunately the effectiveness ofantibiotics has become limited due to an increase in bacterialantibiotic resistance in the face of a decreasing efforts and success indiscovery of new classes of antibiotics. Today, infectious diseases arethe second leading cause of death worldwide and the largest cause ofpremature deaths and loss of work productivity in industrializedcountries. Nosocomial bacterial infections that are resistant to therapyresult in annual costs of more than $2 billion and account for more than80,000 direct and indirect deaths in North America alone, whereas amajor complication of microbial diseases, namely sepsis, accounts for700,000 cases and 140,000 deaths in North America.

A major limitation in antibiotic development has been difficulties infinding new structures with equivalent properties to the conventionalantibiotics, namely low toxicity for the host and a broad spectrum ofaction against bacterial pathogens. Recent novel antibiotic classes,including the oxazolidinones (linezolid), the streptogramins (synercid)and the glycolipids (daptomycin) are all active only against Grampositive pathogens. Cationic antimicrobial peptides, found in mostspecies of life, represent a good template for a new generation ofantimicrobials. They kill both Gram negative and Gram positivemicroorganisms rapidly and directly, do not easily select mutants, workagainst common clinically-resistant bacteria such asmethicillin-resistant Staphylococcus aureus (MRSA) and vancomycinresistant Enterococcus (VRE), show a synergistic effect withconventional antibiotics, and can often activate host innate immunitywithout displaying immunogenicity (Hancock R E W. 2001. Cationicpeptides: effectors in innate immunity and novel antimicrobials. LancetInfectious Diseases 1, 156-164). Moreover, they seem to counteract someof the more harmful aspects of inflammation (e.g. sepsis, endotoxaemia),which is extremely important since rapid killing of bacteria andsubsequent liberation of bacterial components such as LPS orpeptidoglycan can induce fatal immune dysregulation (Jarisch-Herxheimerreaction) (Gough M, Hancock R E W, Kelly N M. 1996. Anti-endotoxicpotential of cationic peptide antimicrobials. Infect. Immun. 64,4922-4927). A need exists in the art for developing new treatments forinfections to be used as broad spectrum antibiotics and/or as agentsthat selectively enhance aspects of innate immunity while suppressingpotentially harmful inflammation.

The innate immune system is a highly effective and evolved generaldefense system that involves a variety of effector functions includingphagocytic cells, complement, and the like, but is generallyincompletely understood. Elements of innate immunity are always presentat low levels and are activated very rapidly when stimulated bypathogens, acting to prevent these pathogens from causing disease.Generally speaking many known innate immune responses are “triggered” bythe binding of microbial signaling molecules, like lipopolysaccharide(LPS), with pattern recognition receptors such as Toll-like receptors(TLR) on the surface of host cells. Many of the effector functions ofinnate immunity are grouped together in the inflammatory response.However, too severe an inflammatory response can result in responsesthat are harmful to the body, and, in an extreme case, sepsis andpotentially death can occur; indeed sepsis occurs in approximately780,000 patients in North America annually with 140,000 deaths. Thus, atherapeutic intervention to boost innate immunity, which is based onstimulation of TLR signaling (for example using a TLR agonist), has thepotential disadvantage that it could stimulate a potentially harmfulinflammatory response and/or exacerbate the natural inflammatoryresponse to infection. A further need exists in the art for therapeuticinverterventions to boost innate immunity that are effective and havefewer undesirable side effects or adverse reactions.

SUMMARY

The invention features antimicrobial and immunomodulatory polypeptides.In some preferred aspects, the polypeptides comprise 7 to 13 aminoacids. Exemplary polypeptides comprise the amino acid sequences of SEQID NOS: 1-969 and 973-1264, and all analogs, homologs, derivatives, andconservative variations thereof. The invention also features additionvariants of these polypeptides, which can comprise up to fiftyadditional amino acids on the amino or carboxy terminal ends of SEQ IDNOS:1-969 and 973-1264, and all analogs, homologs, derivatives, andconservative variations thereof. Where additional amino acids arepresent at the amino and carboxy terminal ends, the amino acids at theamino terminus can be the same as or different from the amino acids atthe carboxy terminus. Polynucleotides encoding the inventivepolypeptides are also provided

Also featured are polypeptides having the sequence X₁-RIRVAV-X₂,X₁-WKWPWWPW-X₂, or X₁-KIWVIRWWR-X₂, or functional variants or mimeticsthereof, wherein X₁ and X₂ independently of one another are 0-5additional amino acids. X₁ and X₂ can, but need not be, identical.

The invention further provides methods for inhibiting the growth ofbacteria cells. The methods generally comprise contacting bacteria withan effective amount of at least one polypeptide having SEQ ID NOS: 1-969and 973-1012, or analogs, derivaties, amidated variations orconservative variations thereof. Polypeptides having the sequenceX₁-RIRVAV-X₂, X₁-WKWPWWPW-X₂, or X₁-KIWVIRWWR-X₂, or functional variantsor mimetics thereof can also be used in the inventive methods. Thepolypeptide can be preset as part of a composition. The bacteria can bea Gram negative bacterium, such as Pseudomonas aeruginosa, Escherichiacoli, or Salmonella enteritidis ssp Typhimurium. The bacteria can be aGram positive bacterium, such as Staphylococcus aureus, Staphylococcusepidermidis, or Enterococcus faecaelis. The methods can, in someaspects, further comprise contacting the bacteria with at least oneantibiotic or lysozyme. The at least one antibiotic or lysozyme can becontacted to the bacteria before, after, or contemporaneously with thepolypeptide or polypeptide composition.

Also featured in accordance with the present invention are methods forenhancing innate immunity. The methods generally comprise contacting acell that expresses at least one polypeptide involved in innate immunitywith an effective amount of a composition comprising at least onepolypeptide having SEQ ID NOS: 1-969 and 973-1012, or analogs,derivaties, amidated variations or conservative variations thereof.Polypeptides having the sequence X₁-RIRVAV-X₂, X₁-WKWPWWPW-X₂, orX₁-KIWVIRWWR-X₂, or functional variants or mimetics thereof can also beused in these inventive methods. Contacting the cell with thecomposition modulates, for example inhibits or enhances, the expressionof the at least one polypeptide involved in innate immunity. Thepolypeptide involved in innate immunity can a chemokine or cytokine. Thepolypeptide involved in innate immunity can be encoded by the geneMCP-1, MCP-3, IL-8, or Gro-α.

The invention also features methods for suppressing a pro-inflammatoryresponse. The methods generally comprise contacting a cell thatexpresses at least one pro-inflammatory cytokine, mediator or protein inresponse to a pro-inflammatory stimulus with an effective amount of acompostion comprising at least one polypeptide having SEQ ID NOS: 1-969and 973-1012, or analogs, derivaties, amidated variations orconservative variations thereof. Polypeptides having the sequenceX₁-RIRVAV-X₂, X₁-WKWPWWPW-X₂, or X₁-KIWVIRWWR-X₂, or functional variantsor mimetics thereof can also be used in these inventive methods.Contacting the cell with the composition inhibits the expression of theat least one pro-inflammatory cytokine, mediator, or protein. In someaspects, the composition inhibits the inflammatory or septic response.In some aspects, the composition inhibits the expression of apro-inflammatory gene or molecule in the cell. In highly preferredaspects, the composition inhibits the expression of TNF-α in the cell.The methods are applicable to suppress the pro-inflammatory responseinduced by any stimulus. In preferred aspects, the methods are utilizedto suppress the inflammatory response induced by a microbe or amicrobial ligand acting on a Toll-like receptor. For example, themicrobial ligand can be a bacterial endotoxin or lipopolysaccharide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. General workflow of the QSAR modeling of antimicrobial peptides

FIG. 2. Complete substitution analysis of the peptide VRLRIRVAVIRA. A.Activity. The first two columns give the position (indicated as the rownumber) and the one-letter code sequence of the original peptideindolicidin. The second and third rows give respectively the columnnumber and the amino acids substituted at each amino acid position. Thusfor example the peptide in the upper left hand corner (column 1, row 1)is ARLRIRVAVIRA (HH253) and in the lower right corner (column 10, row12) VRLRIRVAVIRY (HH468). The results presented within each boxrepresent the relative EC₅₀ value, i.e. the concentration resulting in a50% decrease in luminescence relative to the parent peptide whichappears once in each row (e.g. row 1 column 17, row 2 column 14, etc),as determined by treatment of the lux reporter strain H1001 with peptidefor four hours. Results are colour coded as black=superior activity tothe parent peptide indolicidin; dark grey with white lettering=modestlystronger activity than the parent peptide; light grey with blacklettering=similar activity to the parent peptide; white=very littleactivity. I* symbols for no activity, the EC₅₀ could not be determinedsince the curve showed no bottom. B. An index to FIG. 2A showing thesequence identification numbers of each substitution peptide.

FIG. 3. Complete substitution analysis of the bovine host defensepeptide indolicidin. A. Activity The first two columns give the position(indicated as the row number) and the one-letter code sequence of theoriginal peptide indolicidin. The second and third rows giverespectively the column number and the amino acids substituted at eachamino acid position. Thus for example the peptide in the upper left handcorner (column 1, row 1) is ALPWKWPWWPWRR (HH19) and in the lower rightcorner (column 10, row 13) ILPWKWPWWPWRY (1111252). The resultspresented within each box represent the relative EC₅₀ value, i.e. theconcentration resulting in a 50% decrease in luminescence relative tothe parent peptide which appears once in each row (e.g. row 1 column 7,row 2 column 9, etc), as determined by treatment of the lux reporterstrain 111001 with peptide for four hours. Results are colour coded asblack=superior activity to the parent peptide indolicidin; dark greywith white lettering=modestly stronger activity than the parent peptide;light grey with black lettering=similar activity to the parent peptide;white=very little activity. I* symbols for no activity, the EC₅₀ couldnot be determined since the curve showed no bottom. B. An index to FIG.1A showing the sequence identification numbers of each substitutionpeptide.

FIG. 4. Antimicrobial activity of 200 random peptides. All peptides werecategorized into the activity class “less active than the controlpeptide Bac2A or inactive”.

FIG. 5. Occurrence of amino acids in the new semi-random peptidelibrary. The amino acids are given as the one letter code. Afterassessment of antimicrobial activity using the Lux assay, the occurrenceof amino acids in each activity group, except superior (not enoughmembers), was compared to the occurrence of the semi random librarysetting.

FIG. 6. Occurrence (proportion of total) of amino acids in both (firstand second generation) semi-random peptide libraries.

FIG. 7. Antimicrobial activity of 500 semi-random peptides. The peptideswere categorized into activity classes corresponding to theirantimicrobial activity vs. P. aeruginosa in comparison to the controlpeptide Bac2A. Four activity classes were used: “less active thancontrol or inactive”, “similarly active to control”, “more active thancontrol” and “superior activity”. The number of peptides in each classis expressed as a percentage of the total number of peptides in thelibrary.

FIG. 8. Basis for customized SVL scripts for inductive parameters.Customized SVL scripts (a specialized language of the MOE) werecalculated by using the following fundamental equations) for stericeffect parameters R_(S), parameters of inductive influence σ*, inductivepartial charge ΔN, group ‘inductive’ electronegativity χ_(G) andinductive analogues of local (χ_(i), and s_(i)) and global chemicalhardness and softness (η_(i) and s_(i)). Here R is the covalent atomicradii, r—interatomic distance, χ—atomic electronegativity. The variablesindexed with j subscript describe the influence of a singe atom onto agroup G of n atoms (typically the rest of N-atomic molecule) while Gindices designate group (molecular) quantities. The linear character ofequations (1)-(6) makes inductive descriptors readily computable andsuitable for sizable databases and positions them as appropriateparameters for large-scale QSAR models.

FIG. 9. Similar physical properties of the 4 predicted-activity-basedquartiles of peptides. Panel A: Median MIC, against P. aeruginosa PAO1,of known antimicrobial peptides from training sets A and B (measured)and the corresponding median values for 25 experimentally testedpeptides separated into activity quartiles. Panels B-D: Median values ofcharge (Q), hydrophobicity (P) and amphipathicity/hydrophobic moment(HM).

FIG. 10. Ability of new antimicrobial peptides HHC-10 and HHC-36 toprotect mice against Staph aureus infections. Bacterial loads in theperitoneal lavage from individual mice after 24 hours of infection areshown (solid circles). Dead animals were assigned the highest colonyforming unit (CFU) count obtained in the experiment. The solid linerepresents the arithmetic mean for each group.

FIG. 11. The lack of hemolytic activity (at 375 μg/ml) of 20antimicrobial peptides that demonstrated antibacterial activity. Inaddition to this modest effect at this very high concentration there wasno hemolytic activity at 100 μg/ml.

FIG. 12. Assessment of the ability of peptides to suppress P. aeruginosaLPS (10 ng/ml)-stimulated TNFα production in THP1 cells. Presentedresults are the mean values for 4 wells performed on 2 separateoccasions. The x-axis number labeling is the amount of each peptide inμg/ml for the corresponding peptides.

FIG. 13. Suppression of inflammatory responses by peptides.Monocyte/macrophage-like adherent THP-1 cells were stimulated with 10ng/ml P. aeruginosa LPS and the resulting TNFα response measured.Peptides were added at 10, 20 and 50 μg/ml. The observed TNF-release wasmeasured by ELISA and related to the 100% value of the untreated(without peptide) cells.

FIG. 14. Induction of IL8 release by 7.5×10⁵ human PBMC in response totreatment with 20 or 100 μg/ml of different peptides for 24 hours.

FIG. 15. Induction of MCP-1 release by 7.5×10⁵ human PBMC in response totreatment with 20 or 100 μg/ml of different peptides for 24 hours.

FIG. 16. Induction of MCP3 release by 7.5×10⁵ human PBMC in response totreatment with 20 or 100 μg/ml of different peptides for 24 hours.

FIG. 17. Induction of CXCL1 (Gro-α) release by 7.5×10⁵ human PBMC inresponse to treatment with 20 or 100 μg/ml of different peptides for 24hours.

FIG. 18. Protection of mice from S. aureus infections by peptides HH-2and HH-18 compared to negative control peptide HH-17. Mice were treatedwith 1.6×10¹⁰ CFU of S. aureus intraperitoneally. Four hours postinfection they received a dose of 8 mg/kg peptide IP. The infection wasallowed to progress for 4 or 24 hours after which mice were euthanaisedand plate counts of staphylococci surviving in the peritoneum weredetermined. Bacterial loads in the peritoneal lavage from individualmice after 24 hours of infection are shown (solid circles). The solidline represents the arithmetic mean for each group.

FIG. 19. Protection of mice from S. aureus infections by peptide 1002.Mice were treated with 1.6×10¹⁰ CFU of S. aureus intraperitoneally. Fourhours post infection they received a dose of 8 mg/kg peptide IP. Theinfection was allowed to progress for 4 or 24 hours after which micewere euthanaised and plate counts of staphylococci surviving in theperitoneum were determined. Bacterial loads in the peritoneal lavagefrom individual mice after 24 hours of infection are shown (solidcircles). The solid line represents the arithmetic mean for each group.

FIG. 20. Activities of 200 peptides from the 100 k test set. Q1: top of1^(st) quartile; Q2: Top of 2^(nd) Quartile; Q3: Bottom of 3^(rd)Quartile; Q4: Bottom of 4^(th) Quartile. relIC₅₀ is the relative IC₅₀,the ratio of the IC₅₀ for the experimental peptide to the IC₅₀ of Bac2A.Peptides where the highest concentration failed to reduce theluminescence by at least 50% were identified as inactive.

DETAILED DESCRIPTION

A. Introduction

The present invention is based on the discovery that certain peptidesoriginally identified from the small cationic antimicrobial andimmunomodulatory peptides bactenecin and indolicidin have antimicrobialactivity. Exemplary peptides of the invention include peptides havingthe amino acid sequences of SEQ ID NOS: 1-969, 973-1264, and analogs,derivatives, amidated variations and conservative variations thereof.

The invention further provides a bioinformatic method of predicting newpeptides with good antimicrobial activity through the creation of arandom library of peptides with biased amino acid composition based onthe activity spectrum of the most active peptides investigated, and thenapplying a series of Quantitative Structure-Activity Relationship (QSAR)descriptors and utilizing Artifical Intelligence/Machine-learningapproaches to predict further active peptides.

The invention also provides a method of inhibiting the growth ofbacteria including contacting the bacteria with an inhibiting effectiveamount of at least one peptide of the invention alone, or in combinationwith at least one antibiotic. Classes of antibiotics that can be used insynergistic therapy with the peptides of the invention include, but arenot limited to, aminoglycoside, penicillin, cephalosporin,fluoroquinolone, carbapenem, tetracycline and macrolide.

The invention further provides polynucleotides that encode the peptidesof the invention. Exemplary polynucleotides encode peptides having theamino acid sequences of SEQ ID NOS: 1-969, 973-1264, and analogs,derivatives and conservative variations thereof.

The invention further provides a method of identifying an antimicrobialpeptide having 8 to 12 amino acids that is derived from Bac2A andindolicidin. The method includes contacting a test peptide with amicrobe under conditions sufficient for antimicrobial activity, anddetecting a change in growth or proliferation of the microbe as comparedto the growth or proliferation of the microbe prior to contacting withthe test peptide. In one aspect, the peptide is synthesized in amulti-spot format on a solid support. The peptides of the invention willretain antimicrobial activity when cleaved from the solid support orretain activity when still associated with the solid support. Themicrobe can be a Gram negative bacterium, such as Pseudomonasaeruginosa, Escherichia coli, or Salmonella enteritidis ssp Typhimurium.In another aspect, the microbe can be a Gram positive bacterium, such asStaphylococcus aureus, Staphylococcus epidermidis, or Enterococcusfaecaelis. In yet another aspect, the microbe can be a yeast, such asCandida albicans. The detection can include detecting luminescence in amicrotiter plate luminescence reader over time. In this aspect, themicrobe contains a reporter system, such as a bacterial luciferaseconstruct inserted into the chromosome. For example, the bacterialluciferase construct is inserted into the fliC gene in Pseudomonasaeruginosa.

The invention further provides a method of protecting medical devicesfrom colonization with pathogenic bacteria by coating at least onepeptide of the invention on the surface of the medical device.

Cationic host defense peptides (also known as antimicrobial peptides)are crucial molecules in host defense against pathogenic microbechallenge. Their major effects include direct antimicrobial activity(Hancock, R. E. W., and R. Lehrer. 1998. Cationic peptides: a new sourceof antibiotics. Trends in Biotechnology 16: 82-88.), and an ability tomodulate innate immunity (Hancock, R. E. W. and G. Diamond. 2000. Therole of cationic peptides in innate host defenses. Trends inMicrobiology 8:402-410.; Hancock, R. E. W. 2001. Cationic peptides:effectors in innate immunity and novel antimicrobials. Lancet InfectiousDiseases 1:156-164).

The bovine neutrophil cationic peptides bactenecin (also called bovinedodecapeptide) and indolicidin are arguably the smallest naturallyoccurring antimicrobial peptides. Bactenecin (RLCRIVVIRVCR-NH₂) wasdiscovered in bovine neutrophils by Romeo and coworkers in 1988 (RomeoD, Skerlavaj B, Bolognesi M, Gennaro R. 1988. Structure and bactericidalactivity of an antibiotic dodecapeptide purified from bovineneutrophils. J Biol Chem 263, 9573-5). Bactenecin is stabilized by aninternal disulfide bridge. A linear variant Bac2A (RLARIVVIRVAR-NH₂)shows a similar activity against Gram negative bacteria and an improvedactivity against Gram positive bacteria (Wu M, Hancock R E W. 1999.Improved derivatives of bactenecin, a cyclic dodecameric antimicrobialcationic peptide. Antimicrob Agents Chemother 43, 1274-6). TheC-terminally amidated cationic tridecapeptide indolicidin(ILPWKWPWWPWRR-NH₂, MW=1906), was originally isolated from the largecytoplasmic granules of bovine neutrophils (Selsted, M. E., M. J.Novotny, W. L. Morris, Y. Q. Tang, W. Smith and J. S. Cullor. 1992.Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils.J Biol Chem 267:4292-4295). Indolicidin is active against Gram positiveand Gram negative bacteria, viruses, fungal pathogens, and protozoa(Ryge T S, Doisy X, Ifrah D, Olsen J E, and Hansen P R. New indolicidinanalogues with potent antibacterial activity. J Peptide Research64:171-85, 2004). Both Indolicidin and Bac2A, are known to haveimmunomodulatory activities (Bowdish D M, Davidson D J, Scott M G,Hancock R E W. Immunomodulatory activities of small host defensepeptides. Antimicrobial Agents Chemotherapy 49:1727-32, 2005). Theircommon features, small size, linearity and multiple activities makethese peptides ideal candidates for semi-random design methods such asspot peptide synthesis on cellulose membranes.

The field of chemoinformatics involves computer-aided identification ofnew lead structures and their optimization into drug candidates (EngelT. Basic Overview of Chemoinformatics. Journal of Chemical Informationand Modelling, 46:2267-2277, 2006). One of the most broadly usedchemoinformatics approaches is called Quantitative Structure-ActivityRelationship (QSAR) modeling, which seeks to relate structuralcharacteristics of a molecule (known as descriptors) to its measurableproperties, such as biological activity.

The QSAR analysis found a broad application in antimicrobial discovery.In the series of pilot studies we have also utilized a variety of QSARdescriptors in combination with the approaches of the ArtificialIntelligence to successfully predict antimicrobial activity of limitedsets of organic molecules and cationic peptides (Cherkasov A.‘Inductive’ descriptors. 10 successful years in QSAR. CurrentComputer-Aided Drug Design 1:21-42, 2005; Karakoc E, Sahinalp S C, andCherkasov A. Comparative QSAR- and fragments distribution analysis ofdrugs, druglikes, metabolic substances, and antimicrobial compounds.Journal of Chemical Information and Modelling. 46, 2167-2182, 2006;Cherkasov A. Can ‘bacterial-metabolite-likeness’ model improve odds of‘in silico’ antibiotic discovery? Journal of Chemical Information andModelling, 46, 1214-1222, 2006). An overview of the process used ispresented in FIG. 1.

The method of synthesizing an array of peptides in parallel on cellulosesheets was developed by Ronald Frank in 1992 (Frank, R. Spot synthesis:an easy technique for the positionally addressable, parallel chemicalsynthesis on a membrane support Tetrahedron. 1992 48, 9217-9232). Thistechnique was first carried out manually and used for the identificationof antibody epitopes. Now, with the help of pipetting robots, up to 8000peptides can be synthesized on one cellulose sheet (20×30 cm) (Kramer A,Keitel T, Winkler K, Stocklein W, Hohne W, Schneider-Mergener J. 1997.Molecular basis for the binding promiscuity of an anti-p24 (HIV-1)monoclonal antibody. Cell 91, 799-809). Today, the applications of thistechnology include characterizing homodimer interfaces, screening forkinase recognition sites, optimizing protease inhibitors, and screeningfor DNA binding sites of proteins. We previously adapted thismethodology to create a large number of variants through sequencescrambling, truncations and systematic modifications of peptidesequence, and used a luciferase-based screen to investigate theirability to kill Pseudomonas aeruginosa (Hilpert K, Volkmer-Engert R,Walter T, Hancock R E W. High-throughput generation of smallantibacterial peptides with improved activity. Nature Biotech23:1008-1012, 2005). This permitted us to screen hundreds of 12-merpeptides based on the sequence of the bovine analog Bac2A and determineoptimal amino acid substitutions, and using combinations of amino acidsubstitutions to define peptides of both 8 and 12 amino acids in lengththat had excellent broad spectrum antimicrobial activity.

This method for broad screening represents a rapid and efficient methodto investigate antimicrobial peptide activity. It permits a systematicand highly detailed investigation of the determinants of peptideactivity in very small peptides. Previously, attempts to make smallerpeptides tended to create molecules with modest activities or with goodactivities only when measured in dilute medium. In the studies describedhere we have used a combination of sequence scrambling and single aminoacid substitutions to create a wide range of novel peptides. We havealso examined a range of peptides for anti-endotoxic activity andability to induce chemokines in human peripheral blood mononuclear cells(equivalent to protective immunomodulatory activity) and demonstratethat this procedure can be used to optimize 12-mer cationic peptides forthese properties. This then indicates that the peptides have potentialfor modulating immunity.

The present invention adapts this methodology to create a large numberof variants through sequence scrambling, truncations and systematicmodifications of peptide sequence, and uses a luciferase-based screen toinvestigate their ability to kill Pseudomonas aeruginosa. This broadscreening program represents a rapid and efficient method to investigateantimicrobial peptide activity. It has permitted for the first time asystematic and highly detailed investigation of the determinants ofpeptide activity in very small peptides. Previous attempts to makesmaller peptides have tended to create molecules with modest activitiesor with good activities only when measured in dilute medium.

The peptides of the invention retain activities in the typical mediaused to test in vitro antibiotic activity, making them candidates forclinical therapeutic usage. In addition some of the peptides remaineffective when bound to cellulose sheets, indicating that they have hugepotential for use in coating medical devices, including catheters, toprevent them from becoming colonized with pathogenic bacteria.

The invention provides a number of methods, reagents, and compounds thatcan be used for inhibiting microbial infection or growth. It is to beunderstood that this invention is not limited to particular methods,reagents, compounds, compositions, or biological systems, which can, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular aspects only, and isnot intended to be limiting. As used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a peptide” includes a combination of two or morepeptides, and the like.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Antimicrobial” as used herein means that the peptides of the presentinvention inhibit, prevent, or destroy the growth or proliferation ofmicrobes such as bacteria, fungi, viruses, parasites or the like.“Antiviral” as used herein means that the peptides of the presentinvention inhibit, prevent or destroy the growth or proliferation ofviruses or of virally-infected cells. “Anti-tumor” as used herein meansthat the peptides of the present invention may be used to inhibit thegrowth of or destroy tumors. “Antifungal” as used herein means that thepeptides of the present invention may be used to inhibit the growth ofor destroy fungi. “Antiparasite” as used herein means that the peptidesof the present invention inhibit, prevent, or destroy the growth orproliferation of any organism that lives at the expense of a hostorganism.

“Selective enhancement of innate immunity” as used herein means that thepeptides of the invention are able to upregulate, in mammalian cells,genes and molecules that are natural components of the innate immuneresponse and assist in the resolution of infections without excessiveincreases of pro-inflammatory cytokines like TNFα which can causepotentially harmful inflammation and thus stimulate a sepsis reaction ina subject. The peptides do not stimulate a septic reaction, but dostimulate expression of the one or more genes encoding chemokines orinterleukins that attract immune cells including MCP-1, MCP-3, IL8, andCXCL-1. The peptide may also possess anti-sepsis activity including anability to reduce the expression of TNFα in response to bacterialligands like LPS.

The “amino acid” residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem., 243:3557-59, (1969), abbreviations for amino acid residuesare as shown in the following table.

1-Letter 3-Letter Amino Acid Y Tyr L-tyrosine G Gly L-glycine F PheL-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I IleL-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P ProL-proline K Lys L-lysine H His L-histidine Q Gin L-glutamine E GluL-glutamic acid W Trp L-tryptohan R Arg L-arginine D Asp L-aspartic acidN Asn L-asparagine C Cys L-cysteine

It should be noted that all amino acid residue sequences are representedherein by formulae whose left to right orientation is in theconventional direction of amino-terminus to carboxy-terminus.

B. Peptides

The invention provides an isolated peptide with antimicrobial and/orimmunomodulatory activity. Exemplary peptides of the invention have anamino acid sequence including those listed in Table 1, and analogs,derivatives, amidated variations and conservative variations thereof,wherein the peptides have antimicrobial activity. The peptides of theinvention include SEQ ID NOS:1-969 and 973-1264, as well as the broadergroups of peptides having hydrophilic and hydrophobic substitutions, andconservative variations thereof.

“Isolated” when used in reference to a peptide, refers to a peptidesubstantially free of proteins, lipids, nucleic acids, for example, withwhich it might be naturally associated. Those of skill in the art canmake similar substitutions to achieve peptides with greaterantimicrobial activity and a broader host range. For example, theinvention includes the peptides depicted in SEQ ID NOS:1-969 and973-1264, as well as analogs or derivatives thereof, as long as thebioactivity (e.g., antimicrobial) of the peptide remains. Minormodifications of the primary amino acid sequence of the peptides of theinvention may result in peptides that have substantially equivalentactivity as compared to the specific peptides described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the peptides produced by these modifications areincluded herein as long as the biological activity of the originalpeptide still exists.

Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule that would also have utility.For example, amino or carboxy terminal amino acids that may not berequired for biological activity of the particular peptide can beremoved. Peptides of the invention include any analog, homolog, mutant,isomer or derivative of the peptides disclosed in the present invention,so long as the bioactivity as described herein remains. All peptideswere synthesized using L amino acids, however, all D forms of thepeptides can be synthetically produced. In addition, C-terminalderivatives can be produced, such as C-terminal methyl esters andC-terminal amidates, in order to increase the antimicrobial activity ofa peptide of the invention. The peptide can be synthesized such that thesequence is reversed whereby the last amino acid in the sequence becomesthe first amino acid, and the penultimate amino acid becomes the secondamino acid, and so on. It is well known that such reversed peptidesusually have similar antimicrobial activities to the original sequence.

In certain aspects, the peptides of the invention include peptideanalogs and peptide mimetics. Indeed, the peptides of the inventioninclude peptides having any of a variety of different modifications,including those described herein.

Peptide analogs of the invention are generally designed and produced bychemical modifications of a lead peptide, including, e.g., any of theparticular peptides described herein, such as any of the followingsequences disclosed in the tables. The present invention clearlyestablishes that these peptides in their entirety and derivativescreated by modifying any side chains of the constituent amino acids havethe ability to inhibit, prevent, or destroy the growth or proliferationof microbes such as bacteria, fungi, viruses, parasites or the like. Thepresent invention further encompasses polypeptides up to about 50 aminoacids in length that include the amino acid sequences and functionalvariants or peptide mimetics of the sequences described herein.

In another aspect, a peptide of the present invention is apseudopeptide. Pseudopeptides or amide bond surrogates refers topeptides containing chemical modifications of some (or all) of thepeptide bonds. The introduction of amide bond surrogates not onlydecreases peptide degradation but also may significantly modify some ofthe biochemical properties of the peptides, particularly theconformational flexibility and hydrophobicity.

To improve or alter the characteristics of polypeptides of the presentinvention, protein engineering can be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions, or fusion proteins. Such modifiedpolypeptides can show, e.g., increased/decreased biological activity orincreased/decreased stability. In addition, they can be purified inhigher yields and show better solubility than the corresponding naturalpolypeptide, at least under certain purification and storage conditions.Further, the polypeptides of the present invention can be produced asmultimers including dimers, trimers and tetramers. Multimerization canbe facilitated by linkers, introduction of cysteines to permit creationof interchain disulphide bonds, or recombinantly though heterologouspolypeptides such as Fc regions.

It is known in the art that one or more amino acids can be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. See, e.g., Ron, et al., Biol Chem., 268: 2984-2988, 1993.Accordingly, the present invention provides polypeptides having one ormore residues deleted from the amino terminus. Similarly, many examplesof biologically functional C-terminal deletion mutants are known (see,e.g., Dobeli, et al., 1988). Accordingly, the present invention providespolypeptides having one or more residues deleted from the carboxyterminus. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini asdescribed below.

Other mutants in addition to N- and C-terminal deletion forms of theprotein discussed above are included in the present invention. Thus, theinvention further includes variations of the polypeptides which showsubstantial chaperone polypeptide activity. Such mutants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as to have little effecton activity.

There are two main approaches for studying the tolerance of an aminoacid sequence to change, see, Bowie, et al., Science, 247: 1306-1310,1994. The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality. These studies haverevealed that proteins are surprisingly tolerant of amino acidsubstitutions.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Phe;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. Thus, the polypeptide of the presentinvention can be, for example: (i) one in which one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue can or cannot be one encoded by thegenetic code; or (ii) one in which one or more of the amino acidresidues includes a substituent group; or (iii) one in which thepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol); or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the above form of the polypeptide or a pro-proteinsequence.

Thus, the polypeptides of the present invention can include one or moreamino acid substitutions, deletions, or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein. Thefollowing groups of amino acids represent equivalent changes: (1) Ala,Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val,Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

Furthermore, polypeptides of the present invention can include one ormore amino acid substitutions that mimic modified amino acids. Anexample of this type of substitution includes replacing amino acids thatare capable of being phosphorylated (e.g., serine, threonine, ortyrosine) with a negatively charged amino acid that resembles thenegative charge of the phosphorylated amino acid (e.g., aspartic acid orglutamic acid). Also included is substitution of amino acids that arecapable of being modified by hydrophobic groups (e.g., arginine) withamino acids carrying bulky hydrophobic side chains, such as tryptophanor phenylalanine. Therefore, a specific aspect of the invention includespolypeptides that include one or more amino acid substitutions thatmimic modified amino acids at positions where amino acids that arecapable of being modified are normally positioned. Further included arepolypeptides where any subset of modifiable amino acids is substituted.For example, a polypeptide that includes three serine residues can besubstituted at any one, any two, or all three of said serines.Furthermore, any polypeptide amino acid capable of being modified can beexcluded from substitution with a modification-mimicking amino acid.

The present invention is further directed to fragments of thepolypeptides of the present invention. More specifically, the presentinvention embodies purified, isolated, and recombinant polypeptidescomprising at least any one integer between 6 and 504 (or the length ofthe polypeptides amino acid residues minus 1 if the length is less than1000) of consecutive amino acid residues. Preferably, the fragments areat least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25,30, 35, 40, 50 or more consecutive amino acids of a polypeptide of thepresent invention.

The present invention also provides for the exclusion of any species ofpolypeptide fragments of the present invention specified by 5′ and 3′positions or sub-genuses of polypeptides specified by size in aminoacids as described above. Any number of fragments specified by 5′ and 3′positions or by size in amino acids, as described above, can beexcluded.

In addition, it should be understood that in certain aspects, thepeptides of the present invention include two or more modifications,including, but not limited to those described herein. By taking into theaccount the features of the peptide drugs on the market or under currentdevelopment, it is clear that most of the peptides successfullystabilized against proteolysis consist of a mixture of several types ofthe above described modifications. This conclusion is understood in thelight of the knowledge that many different enzymes are implicated inpeptide degradation.

C. Peptides, Peptide Variants, and Peptide Mimetics

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but which functions in a mannersimilar to a naturally occurring amino acid. Non-natural residues arewell described in the scientific and patent literature; a few exemplarynon-natural compositions useful as mimetics of natural amino acidresidues and guidelines are described below. Mimetics of aromatic aminoacids can be generated by replacing by, e.g., D- or L-naphylalanine; D-or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings(See also, table entitled “Non-Natural Amino Acids” in Appendix below).

“Peptide” as used herein includes peptides that are conservativevariations of those peptides specifically exemplified herein.“Conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include, but are not limited to, thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan,tyrosine, norleucine or methionine for another, or the substitution ofone polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. Neutral hydrophilic amino acids that can be substituted forone another include asparagine, glutamine, serine and threonine. Theterm “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Such conservative substitutions arewithin the definition of the classes of the peptides of the invention.“Cationic” as is used to refer to any peptide that possesses sufficientpositively charged amino acids to have a pI (isoelectric point) greaterthan about 9.0.

The biological activity of the peptides can be determined by standardmethods known to those of skill in the art, such as “minimal inhibitoryconcentration (MIC)” assay described in the present examples, wherebythe lowest concentration at which no change in OD is observed for agiven period of time is recorded as MIC.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound that hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, a mimeticcomposition is within the scope of the invention if, when administeredto or expressed in a cell, e.g., a polypeptide fragment of ananimicrobial protein having antimicrobial activity.

Polypeptide mimetic compositions can contain any combination ofnon-natural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, i.e., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like. For example, a polypeptide can becharacterized as a mimetic when all or some of its residues are joinedby chemical means other than natural peptide bonds. Individualpeptidomimetic residues can be joined by peptide bonds, other chemicalbonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono) alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholin-yl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution with,e.g., (in addition to lysine and arginine) the amino acids ornithine, orcitrulline. Asparaginyl and glutaminyl residues can be deaminated to thecorresponding aspartyl or glutamyl residues.

Arginine residue mimetics can be generated by reacting arginyl with,e.g., one or more conventional reagents, including, e.g., phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably underalkaline conditions. Tyrosine residue mimetics can be generated byreacting tyrosyl with, e.g., aromatic diazonium compounds ortetranitromethane. N-acetylimidizol and tetranitromethane can be used toform O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Histidineresidue mimetics can be generated by reacting histidyl with, e.g.,diethylprocarbonate or para-bromophenacyl bromide. Other mimeticsinclude, e.g., those generated by hydroxylation of lysine;phosphorylation of the hydroxyl groups of seryl or threonyl residues;methylation of the alpha-amino groups of lysine, arginine and histidine;acetylation of the N-terminal amine; methylation of main chain amideresidues or substitution with N-methyl amino acids; or amidation ofC-terminal carboxyl groups.

A component of a polypeptide of the invention can also be replaced by anamino acid (or peptidomimetic residue) of the opposite chirality. Thus,any amino acid naturally occurring in the L-configuration (which canalso be referred to as the R or S, depending upon the structure of thechemical entity) can be replaced with the amino acid of the samechemical structural type or a peptidomimetic, but of the oppositechirality, referred to as the D-amino acid, but which can additionallybe referred to as the R- or S-form

The invention also provides polypeptides that are “substantiallyidentical” to an exemplary polypeptide of the invention. A“substantially identical” amino acid sequence is a sequence that differsfrom a reference sequence by one or more conservative ornon-conservative amino acid substitutions, deletions, or insertions,particularly when such a substitution occurs at a site that is not theactive site of the molecule, and provided that the polypeptideessentially retains its functional properties. A conservative amino acidsubstitution, for example, substitutes one amino acid for another of thesame class (e.g., substitution of one hydrophobic amino acid, such asisoleucine, valine, leucine, or methionine, for another, or substitutionof one polar amino acid for another, such as substitution of argininefor lysine, glutamic acid for aspartic acid or glutamine forasparagine). One or more amino acids can be deleted, for example, froman antimicrobial polypeptide having antimicrobial activity of theinvention, resulting in modification of the structure of thepolypeptide, without significantly altering its biological activity. Forexample, amino- or carboxyl-terminal, or internal, amino acids that arenot required for antimicrobial activity can be removed.

The skilled artisan will recognize that individual synthetic residuesand polypeptides incorporating these mimetics can be synthesized using avariety of procedures and methodologies, which are well described in thescientific and patent literature, e.g., Organic Syntheses CollectiveVolumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Peptides andpeptide mimetics of the invention can also be synthesized usingcombinatorial methodologies. Various techniques for generation ofpeptide and peptidomimetic libraries are well known, and include, e.g.,multipin, tea bag, and split-couple-mix techniques; see, e.g.,al-Obeidi, Mol. Biotechnol. 9: 205-223, 1998; Hruby, Curr. Opin. Chem.Biol. 1: 114-119, 1997; Ostergaard, Mol. Divers. 3: 17-27, 1997;Ostresh, Methods Enzymol. 267: 220-234, 1996. Modified peptides of theinvention can be further produced by chemical modification methods, see,e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, FreeRadic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33:7886-7896, 1994.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers, Nucleic Acids Res.Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser. 225-232,1980; Banga, Therapeutic Peptides and Proteins, Formulation, Processingand Delivery Systems Technomic Publishing Co., Lancaster, Pa., 1995. Forexample, peptide synthesis can be performed using various solid-phasetechniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield,Methods Enzymol. 289: 3-13, 1997) and automated synthesis can beachieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) inaccordance with the instructions provided by the manufacturer.

Peptides of the invention can be synthesized by such commonly usedmethods as t-BOC or FMOC protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C terminus of the peptide (See, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods described in Merrifield, J. Am.Chem. Soc., 85:2149, (1962), and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

Analogs, polypeptide fragment of antimicrobial protein havingantimicrobial activity, are generally designed and produced by chemicalmodifications of a lead peptide, including, e.g., any of the particularpeptides described herein, such as any of the sequences including SEQ IDNOS:1-969 and 973-1264.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 66%, 67%, 68%, 69%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region (e.g., nucleotide sequence encoding anantibody described herein or amino acid sequence of an antibodydescribed herein), when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This term alsorefers to, or can be applied to, the compliment of a test sequence. Theterm also includes sequences that have deletions and/or additions, aswell as those that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence can be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970,by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85: 2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al., eds. 1995 supplement).

Programs for searching for alignments are well known in the art, e.g.,BLAST and the like. For example, if the target species is human, asource of such amino acid sequences or gene sequences (germline orrearranged antibody sequences) can be found in any suitable referencedatabase such as Genbank, the NCBI protein databank (found on the web atthe site: ncbi.nlm.nih.gov/BLAST), VBASE, a database of human antibodygenes

(found on the web at the site: mrc-cpe.cam.ac.uk/imt-doc), and the Kabatdatabase of immunoglobulins

(found on the web at the site: immuno.bme.nwu.edu) or translatedproducts thereof. If the alignments are done based on the nucleotidesequences, then the selected genes should be analyzed to determine whichgenes of that subset have the closest amino acid homology to theoriginating species antibody. It is contemplated that amino acidsequences or gene sequences which approach a higher degree homology ascompared to other sequences in the database can be utilized andmanipulated in accordance with the procedures described herein.Moreover, amino acid sequences or genes which have lesser homology canbe utilized when they encode products which, when manipulated andselected in accordance with the procedures described herein, exhibitspecificity for the predetermined target antigen. In certain aspects, anacceptable range of homology is greater than about 50%. It should beunderstood that target species can be other than human.

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25: 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (found on the web at the site:ncbi.nlm.nih.gov) . This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length (W)in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold. These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare extended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915,1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

D. Polypeptides and Functional Variants Thereof

“Polypeptide” includes proteins, fusion proteins, oligopeptides andpolypeptide derivatives, with the exception that peptidomimetics areconsidered to be small molecules herein.

A “protein” is a molecule having a sequence of amino acids that arelinked to each other in a linear molecule by peptide bonds. The termprotein refers to a polypeptide that is isolated from a natural source,or produced from an isolated cDNA using recombinant DNA technology; andhas a sequence of amino acids having a length of at least about 200amino acids.

A “fusion protein” is a type of recombinant protein that has an aminoacid sequence that results from the linkage of the amino acid sequencesof two or more normally separate polypeptides.

A “protein fragment” is a proteolytic fragment of a larger polypeptide,which may be a protein or a fusion protein. A proteolytic fragment maybe prepared by in vivo or in vitro proteolytic cleavage of a largerpolypeptide, and is generally too large to be prepared by chemicalsynthesis. Proteolytic fragments have amino acid sequences having alength from about 200 to about 1,000 amino acids.

An “oligopeptide” or “peptide” is a polypeptide having a short aminoacid sequence (i.e., 2 to about 200 amino acids). An oligopeptide isgenerally prepared by chemical synthesis.

Although oligopeptides and protein fragments may be otherwise prepared,it is possible to use recombinant DNA technology and/or in vitrobiochemical manipulations. For example, a nucleic acid encoding an aminoacid sequence may be prepared and used as a template for in vitrotranscription/translation reactions. In such reactions, an exogenousnucleic acid encoding a preselected polypeptide is introduced into amixture that is essentially depleted of exogenous nucleic acids thatcontains all of the cellular components required for transcription andtranslation. One or more radiolabeled amino acids are added before orwith the exogenous DNA, and transcription and translation are allowed toproceed. Because the only nucleic acid present in the reaction mix isthe exogenous nucleic acid added to the reaction, only polypeptidesencoded thereby are produced, and incorporate the radiolabeled aminoacid(s). In this manner, polypeptides encoded by a preselected exogenousnucleic acid are radiolabeled. Although other proteins are present inthe reaction mix, the preselected polypeptide is the only one that isproduced in the presence of the radiolabeled amino acids and is thusuniquely labeled.

As is explained in detail below, “polypeptide derivatives” includewithout limitation mutant polypeptides, chemically modifiedpolypeptides, and peptidomimetics.

The polypeptides of this invention, including the analogs and othermodified variants, may generally be prepared following known techniques.Preferably, synthetic production of the polypeptide of the invention maybe according to the solid phase synthetic method. For example, the solidphase synthesis is well understood and is a common method forpreparation of polypeptides, as are a variety of modifications of thattechnique. Merrifield, J. Am. Chem. Soc., 85: 2149, 1964; Stewart andYoung, Solid Phase polypeptide Synthesis, Pierce Chemical Company,Rockford, Ill., 1984; Bodanszky and Bodanszky, The Practice ofpolypeptide Synthesis, Springer-Verlag, New York, 1984; Atherton andSheppard, Solid Phase polypeptide Synthesis: A Practical Approach, IRLPress, New York, 1989. See, also, the specific method described inExample 1 below.

Alternatively, polypeptides of this invention may be prepared inrecombinant systems using polynucleotide sequences encoding thepolypeptides.

A “variant” or “functional variant” of a polypeptide is a compound thatis not, by definition, a polypeptide, i.e., it contains at least onechemical linkage that is not a peptide bond. Thus, polypeptidederivatives include without limitation proteins that naturally undergopost-translational modifications such as, e.g., glycosylation. It isunderstood that a polypeptide of the invention may contain more than oneof the following modifications within the same polypeptide. Preferredpolypeptide derivatives retain a desirable attribute, which may bebiological activity; more preferably, a polypeptide derivative isenhanced with regard to one or more desirable attributes, or has one ormore desirable attributes not found in the parent polypeptide. Althoughthey are described in this section, peptidomimetics are taken as smallmolecules in the present disclosure.

A polypeptide having an amino acid sequence identical to that found in aprotein prepared from a natural source is a “wildtype” polypeptide.Functional variants of polypeptides can be prepared by chemicalsynthesis, including without limitation combinatorial synthesis.

Functional variants of polypeptides larger than oligopeptides can beprepared using recombinant DNA technology by altering the nucleotidesequence of a nucleic acid encoding a polypeptide. Although somealterations in the nucleotide sequence will not alter the amino acidsequence of the polypeptide encoded thereby (“silent” mutations), manywill result in a polypeptide having an altered amino acid sequence thatis altered relative to the parent sequence. Such altered amino acidsequences may comprise substitutions, deletions and additions of aminoacids, with the proviso that such amino acids are naturally occurringamino acids.

Thus, subjecting a nucleic acid that encodes a polypeptide tomutagenesis is one technique that can be used to prepare Functionalvariants of polypeptides, particularly ones having substitutions ofamino acids but no deletions or insertions thereof. A variety ofmutagenic techniques are known that can be used in vitro or in vivoincluding without limitation chemical mutagenesis and PCR-mediatedmutagenesis. Such mutagenesis may be randomly targeted (i.e., mutationsmay occur anywhere within the nucleic acid) or directed to a section ofthe nucleic acid that encodes a stretch of amino acids of particularinterest. Using such techniques, it is possible to prepare randomized,combinatorial or focused compound libraries, pools and mixtures.

Polypeptides having deletions or insertions of naturally occurring aminoacids may be synthetic oligopeptides that result from the chemicalsynthesis of amino acid sequences that are based on the amino acidsequence of a parent polypeptide but which have one or more amino acidsinserted or deleted relative to the sequence of the parent polypeptide.Insertions and deletions of amino acid residues in polypeptides havinglonger amino acid sequences may be prepared by directed mutagenesis.

As contemplated by this invention, “polypeptide” includes those havingone or more chemical modification relative to another polypeptide, i.e.,chemically modified polypeptides. The polypeptide from which achemically modified polypeptide is derived may be a wildtype protein, afunctional variant protein or a functional variant polypeptide, orpolypeptide fragments thereof; an antibody or other polypeptide ligandaccording to the invention including without limitation single-chainantibodies, crystalline proteins and polypeptide derivatives thereof; orpolypeptide ligands prepared according to the disclosure. Preferably,the chemical modification(s) confer(s) or improve(s) desirableattributes of the polypeptide but does not substantially alter orcompromise the biological activity thereof. Desirable attributes includebut are limited to increased shelf-life; enhanced serum or other in vivostability; resistance to proteases; and the like. Such modificationsinclude by way of non-limiting example N-terminal acetylation,glycosylation, and biotinylation.

An effective approach to confer resistance to peptidases acting on theN-terminal or C-terminal residues of a polypeptide is to add chemicalgroups at the polypeptide termini, such that the modified polypeptide isno longer a substrate for the peptidase. One such chemical modificationis glycosylation of the polypeptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of polypeptides in human serum(Powell et al., Pharma. Res. 10: 1268-1273, 1993). Other chemicalmodifications which enhance serum stability include, but are not limitedto, the addition of an N-terminal alkyl group, consisting of a loweralkyl of from 1 to 20 carbons, such as an acetyl group, and/or theaddition of a C-terminal amide or substituted amide group.

The presence of an N-terminal D-amino acid increases the serum stabilityof a polypeptide that otherwise contains L-amino acids, becauseexopeptidases acting on the N-terminal residue cannot utilize a D-aminoacid as a substrate. Similarly, the presence of a C-terminal D-aminoacid also stabilizes a polypeptide, because serum exopeptidases actingon the C-terminal residue cannot utilize a D-amino acid as a substrate.With the exception of these terminal modifications, the amino acidsequences of polypeptides with N-terminal and/or C-terminal D-aminoacids are usually identical to the sequences of the parent L-amino acidpolypeptide.

Substitution of unnatural amino acids for natural amino acids in asubsequence of a polypeptide can confer or enhance desirable attributesincluding biological activity. Such a substitution can, for example,confer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of polypeptides with unnatural amino acids isroutine and known in the art (see, for example, Coller, et al. 1993,cited above).

Different host cells will contain different post-translationalmodification mechanisms that may provide particular types ofpost-translational modification of a fusion protein if the amino acidsequences required for such modifications is present in the fusionprotein. A large number (about 100) of post-translational modificationshave been described, a few of which are discussed herein. One skilled inthe art will be able to choose appropriate host cells, and designchimeric genes that encode protein members comprising the amino acidsequence needed for a particular type of modification.

Glycosylation is one type of post-translational chemical modificationthat occurs in many eukaryotic systems, and may influence the activity,stability, pharmacogenetics, immunogenicity and/or antigenicity ofproteins. However, specific amino acids must be present at such sites torecruit the appropriate glycosylation machinery, and not all host cellshave the appropriate molecular machinery. Saccharomyces cerevisieae andPichia pastoris provide for the production of glycosylated proteins, asdo expression systems that utilize insect cells, although the pattern ofglyscoylation may vary depending on which host cells are used to producethe fusion protein.

Another type of post-translation modification is the phosphorylation ofa free hydroxyl group of the side chain of one or more Ser, Thr or Tyrresidues, Protein kinases catalyze such reactions. Phosphorylation isoften reversible due to the action of a protein phosphatase, an enzymethat catalyzes the dephosphorylation of amino acid residues.

Differences in the chemical structure of amino terminal residues resultfrom different host cells, each of which may have a different chemicalversion of the methionine residue encoded by a start codon, and thesewill result in amino termini with different chemical modifications.

For example, many or most bacterial proteins are synthesized with anamino terminal amino acid that is a modified form of methionine, i.e.,N-formyl-methionine (fMet). Although the statement is often made thatall bacterial proteins are synthesized with an fMet initiator aminoacid; although this may be true for E. coli, recent studies have shownthat it is not true in the case of other bacteria such as Pseudomonasaeruginosa (Newton et al., J. Biol. Chem. 274: 22143-22146, 1999). Inany event, in E. coli, the formyl group of fMet is usually enzymaticallyremoved after translation to yield an amino terminal methionine residue,although the entire fMet residue is sometimes removed (see Hershey,Chapter 40, “Protein Synthesis” in: Escherichia coli and Salmonellatyphimurium: Cellular and Molecular Biology, Neidhardt, Frederick C.,Editor in Chief, American Society for Microbiology, Washington, D.C.,1987, Volume 1, pages 613-647, and references cited therein.). E. colimutants that lack the enzymes (such as, e.g., formylase) that catalyzesuch post-translational modifications will produce proteins having anamino terminal fMet residue (Guillon et al., J. Bacteriol. 174:4294-4301, 1992).

In eukaryotes, acetylation of the initiator methionine residue, or thepenultimate residue if the initiator methionine has been removed,typically occurs co- or post-translationally. The acetylation reactionsare catalyzed by N-terminal acetyltransferases (NATs, a.k.a.N-alpha-acetyltransferases), whereas removal of the initiator methionineresidue is catalyzed by methionine aminopeptidases (for reviews, seeBradshaw et al., Trends Biochem. Sci. 23: 263-267, 1998; and Driessen etal., CRC Crit. Rev. Biochem. 18: 281-325, 1985). Amino terminallyacetylated proteins are said to be “N-acetylated,” “N alpha acetylated”or simply “acetylated.”

Another post-translational process that occurs in eukaryotes is thealpha-amidation of the carboxy terminus. For reviews, see Eipper et al.Annu. Rev. Physiol. 50: 333-344, 1988, and Bradbury et al. Lung Cancer14: 239-251, 1996. About 50% of known endocrine and neuroendocrinepeptide hormones are alpha-amidated (Treston et al., Cell Growth Differ.4: 911-920, 1993). In most cases, carboxy alpha-amidation is required toactivate these peptide hormones.

E. Polypeptide Mimetic

In general, a polypeptide mimetic (“peptidomimetic”) is a molecule thatmimics the biological activity of a polypeptide but is no longerpeptidic in chemical nature. By strict definition, a peptidomimetic is amolecule that contains no peptide bonds (that is, amide bonds betweenamino acids). However, the term peptidomimetic is sometimes used todescribe molecules that are no longer completely peptidic in nature,such as pseudo-peptides, semi-peptides and peptoids. Examples of somepeptidomimetics by the broader definition (where part of a polypeptideis replaced by a structure lacking peptide bonds) are described below.Whether completely or partially non-peptide, peptidomimetics accordingto this invention provide a spatial arrangement of reactive chemicalmoieties that closely resembles the three-dimensional arrangement ofactive groups in the polypeptide on which the peptidomimetic is based.As a result of this similar active-site geometry, the peptidomimetic haseffects on biological systems that are similar to the biologicalactivity of the polypeptide.

There are several potential advantages for using a mimetic of a givenpolypeptide rather than the polypeptide itself. For example,polypeptides may exhibit two undesirable attributes, i.e., poorbioavailability and short duration of action. Peptidomimetics are oftensmall enough to be both orally active and to have a long duration ofaction. There are also problems associated with stability, storage andimmunoreactivity for polypeptides that are not experienced withpeptidomimetics.

Candidate, lead and other polypeptides having a desired biologicalactivity can be used in the development of peptidomimetics with similarbiological activities. Techniques of developing peptidomimetics frompolypeptides are known. Peptide bonds can be replaced by non-peptidebonds that allow the peptidomimetic to adopt a similar structure, andtherefore biological activity, to the original polypeptide. Furthermodifications can also be made by replacing chemical groups of the aminoacids with other chemical groups of similar structure. The developmentof peptidomimetics can be aided by determining the tertiary structure ofthe original polypeptide, either free or bound to a ligand, by NMRspectroscopy, crystallography and/or computer-aided molecular modeling.These techniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal polypeptide (Dean, BioEssays, 16: 683-687, 1994; Cohen andShatzmiller, J. Mol. Graph., 11: 166-173, 1993; Wiley and Rich, Med.Res. Rev., 13: 327-384, 1993; Moore, Trends Pharmacol. Sci., 15:124-129, 1994; Hruby, Biopolymers, 33: 1073-1082, 1993; Bugg et al.,Sci. Am., 269: 92-98, 1993, all incorporated herein by reference).

Thus, through use of the methods described above, the present inventionprovides compounds exhibiting enhanced therapeutic activity incomparison to the polypeptides described above. The peptidomimeticcompounds obtained by the above methods, having the biological activityof the above named polypeptides and similar three-dimensional structure,are encompassed by this invention. It will be readily apparent to oneskilled in the art that a peptidomimetic can be generated from any ofthe modified polypeptides described in the previous section or from apolypeptide bearing more than one of the modifications described fromthe previous section. It will furthermore be apparent that thepeptidomimetics of this invention can be further used for thedevelopment of even more potent non-peptidic compounds, in addition totheir utility as therapeutic compounds.

Specific examples of peptidomimetics derived from the polypeptidesdescribed in the previous section are presented below. These examplesare illustrative and not limiting in terms of the other or additionalmodifications.

Proteases act on peptide bonds. It therefore follows that substitutionof peptide bonds by pseudopeptide bonds confers resistance toproteolysis. A number of pseudopeptide bonds have been described that ingeneral do not affect polypeptide structure and biological activity. Thereduced isostere pseudopeptide bond is a suitable pseudopeptide bondthat is known to enhance stability to enzymatic cleavage with no orlittle loss of biological activity (Couder, et al., Int. J. PolypeptideProtein Res. 41: 181-184, 1993, incorporated herein by reference). Thus,the amino acid sequences of these compounds may be identical to thesequences of their parent L-amino acid polypeptides, except that one ormore of the peptide bonds are replaced by an isosteric pseudopeptidebond. Preferably the most N-terminal peptide bond is substituted, sincesuch a substitution would confer resistance to proteolysis byexopeptidases acting on the N-terminus.

To confer resistance to proteolysis, peptide bonds may also besubstituted by retro-inverso pseudopeptide bonds (Dalpozzo, et al., Int.J. Polypeptide Protein Res. 41: 561-566, incorporated herein byreference). According to this modification, the amino acid sequences ofthe compounds may be identical to the sequences of their L-amino acidparent polypeptides, except that one or more of the peptide bonds arereplaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus.

Peptoid derivatives of polypeptides represent another form of modifiedpolypeptides that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., Proc. Natl. Acad. Sci. USA,89: 9367-9371, 1992, and incorporated herein by reference). Peptoids areoligomers of N-substituted glycines. A number of N-alkyl groups havebeen described, each corresponding to the side chain of a natural aminoacid.

F. Polynucleotides

The invention includes polynucleotides encoding peptides of theinvention. Exemplary polynucleotides encode peptides including thoselisted in Table 1, and analogs, derivatives, amidated variations andconservative variations thereof, wherein the peptides have antimicrobialactivity. The peptides of the invention include SEQ ID NOS:1-969 and973-1264, as well as the broader groups of peptides having hydrophilicand hydrophobic substitutions, and conservative variations thereof.

“Isolated” when used in reference to a polynucleotide, refers to apolynucleotide substantially free of proteins, lipids, nucleic acids,for example, with which it is naturally associated. As used herein,“polynucleotide” refers to a polymer of deoxyribonucleotides orribonucleotides, in the form of a separate fragment or as a component ofa larger construct. DNA encoding a peptide of the invention can beassembled from cDNA fragments or from oligonucleotides which provide asynthetic gene which is capable of being expressed in a recombinanttranscriptional unit. Polynucleotide sequences of the invention includeDNA, RNA and cDNA sequences. A polynucleotide sequence can be deducedfrom the genetic code, however, the degeneracy of the code must be takeninto account. Polynucleotides of the invention include sequences whichare degenerate as a result of the genetic code. Such polynucleotides areuseful for the recombinant production of large quantities of a peptideof interest, such as the peptide of SEQ ID NOS:1-969 and 973-1264.

In the present invention, the polynucleotides encoding the peptides ofthe invention may be inserted into a recombinant “expression vector”.The term “expression vector” refers to a plasmid, virus or other vehicleknown in the art that has been manipulated by insertion or incorporationof genetic sequences. Such expression vectors of the invention arepreferably plasmids that contain a promoter sequence that facilitatesthe efficient transcription of the inserted genetic sequence in thehost. The expression vector typically contains an origin of replication,a promoter, as well as specific genes that allow phenotypic selection ofthe transformed cells. For example, the expression of the peptides ofthe invention can be placed under control of E. coli chromosomal DNAcomprising a lactose or lac operon which mediates lactose utilization byelaborating the enzyme beta-galactosidase. The lac control system can beinduced by IPTG. A plasmid can be constructed to contain the lac Iqrepressor gene, permitting repression of the lac promoter until IPTG isadded. Other promoter systems known in the art include beta-lactamase,lambda promoters, the protein A promoter, and the tryptophan promotersystems. While these are the most commonly used, other microbialpromoters, both inducible and constitutive, can be utilized as well. Thevector contains a replicon site and control sequences which are derivedfrom species compatible with the host cell. In addition, the vector maycarry specific gene(s) which are capable of providing phenotypicselection in transformed cells. For example, the beta-lactamase geneconfers ampicillin resistance to those transformed cells containing thevector with the beta-lactamase gene. An exemplary expression system forproduction of the peptides of the invention is described in U.S. Pat.No. 5,707,855.

Transformation of a host cell with the polynucleotide may be carried outby conventional techniques known to those skilled in the art. Forexample, where the host is prokaryotic, such as E. coli, competent cellsthat are capable of DNA uptake can be prepared from cells harvestedafter exponential growth and subsequently treated by the CaCl₂ methodusing procedures known in the art. Alternatively, MgCl₂ or RbCl could beused.

In addition to conventional chemical methods of transformation, theplasmid vectors of the invention may be introduced into a host cell byphysical means, such as by electroporation or microinjection.Electroporation allows transfer of the vector by high voltage electricimpulse, which creates pores in the plasma membrane of the host and isperformed according to methods known in the art. Additionally, clonedDNA can be introduced into host cells by protoplast fusion, usingmethods known in the art.

DNA sequences encoding the peptides can be expressed in vivo by DNAtransfer into a suitable host cell. “Host cells” of the invention arethose in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that not all progeny are identical to the parental cell,since there may be mutations that occur during replication. However,such progeny are included when the terms above are used. Preferred hostcells of the invention include E. coli, S. aureus and P. aeruginosa,although other Gram negative and Gram positive organisms known in theart can be utilized as long as the expression vectors contain an originof replication to permit expression in the host.

The polynucleotide sequence encoding the peptide used according to themethod of the invention can be isolated from an organism or synthesizedin the laboratory. Specific DNA sequences encoding the peptide ofinterest can be obtained by: 1) isolation of a double-stranded DNAsequence from the genomic DNA; 2) chemical manufacture of a DNA sequenceto provide the necessary codons for the peptide of interest; and 3) invitro synthesis of a double-stranded DNA sequence by reversetranscription of mRNA isolated from a donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed that isgenerally referred to as cDNA.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired peptideproduct is known. In the present invention, the synthesis of a DNAsequence has the advantage of allowing the incorporation of codons thatare more likely to be recognized by a bacterial host, thereby permittinghigh level expression without difficulties in translation. In addition,virtually any peptide can be synthesized, including those encodingnatural peptides, variants of the same, or synthetic peptides.

When the entire sequence of the desired peptide is not known, the directsynthesis of DNA sequences is not possible and the method of choice isthe formation of cDNA sequences. Among the standard procedures forisolating cDNA sequences of interest is the formation of plasmid orphage containing cDNA libraries that are derived from reversetranscription of mRNA that is abundant in donor cells that have a highlevel of genetic expression. When used in combination with polymerasechain reaction technology, even rare expression products can be cloned.In those cases where significant portions of the amino acid sequence ofthe peptide are known, the production of labeled single ordouble-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single stranded form (Jay et al.,Nuc. Acid Res., 11:2325, 1983).

G. QSAR Descriptors and Machine Learning Methods

The invention further provides a bioinformatic method of predicting newpeptides with good antimicrobial activity through the creation of arandom library of peptides with biased amino acid composition based onthe activity spectrum of the most active peptides investigated, and thenapplying a series of Quantitative Structure-Activity Relationship (QSAR)descriptors and utilizing Artifical Intelligence/Machine-learningapproaches to predict further active peptides.

H. Methods of Use—Direct Antimicrobial

The invention also provides a method of inhibiting the growth ofbacteria including contacting the bacteria with an inhibiting effectiveamount of a peptide of the invention, including SEQ ID NOS:1-969 and973-1264, and analogs, derivatives, amidated variations and conservativevariations thereof, wherein the peptides have antimicrobial activity.

The term “contacting” refers to exposing the bacteria to the peptide sothat the peptide can effectively inhibit, kill, or lyse bacteria, bindendotoxin (LPS), or permeabilize Gram negative bacterial outermembranes. Contacting may be in vitro, for example by adding the peptideto a bacterial culture to test for susceptibility of the bacteria to thepeptide. Contacting may be in vivo, for example administering thepeptide to a subject with a bacterial disorder, such as septic shock orinfection. Contacting may further involve coating an object (e.g.,medical device) such as a catheter to inhibit bacteria with which itcomes into contact, thus preventing it from becoming colonized with thebacteria. “Inhibiting” or “inhibiting effective amount” refers to theamount of peptide that is required to cause a bacteriostatic orbactericidal effect. Examples of bacteria that may be inhibited includeEscherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae,Salmonella enteritidis subspecies Typhimurium, Staphylococcus aureus,Enterococcus facaelis, Listeria monocytogenes, Corynebacterium xerosis,Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mitisand Staphylococcuus epidermidis.

The method of inhibiting the growth of bacteria may further include theaddition of antibiotics for combination or synergistic therapy. Theappropriate antibiotic administered will typically depend on thesusceptibility of the bacteria such as whether the bacteria is Gramnegative or Gram positive, and will be easily discernable by one ofskill in the art. Examples of particular classes of antibiotics usefulfor synergistic therapy with the peptides of the invention includeaminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin),cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g.,ciprofloxacin), carbapenems (e.g., imipenem), tetracyclines andmacrolides (e.g., erythromycin and clarithromycin). The method ofinhibiting the growth of bacteria may further include the addition ofantibiotics for combination or synergistic therapy. The appropriateantibiotic administered will typically depend on the susceptibility ofthe bacteria such as whether the bacteria is Gram negative or Grampositive, and will be easily discernable by one of skill in the art.Further to the antibiotics listed above, typical antibiotics includeaminoglycosides (amikacin, gentamicin, kanamycin, netilmicin,t-obramycin, streptomycin), macrolides (azithromycin, clarithromycin,erythromycin, erythromycinestolate/ethylsuccinate/gluceptate/lactobionate/stearate), beta-lactamssuch as penicillins (e.g., penicillin G, penicillin V, methicillin,nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin,amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin andpiperacillin), or cephalosporins (e.g., cephalothin, cefazolin,cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole,cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime,ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime,and cefsulodin) or carbapenems (e.g., imipenem, meropenem, panipenem),or monobactams (e.g., aztreonam). Other classes of antibiotics includequinolones (e.g., fleroxacin, nalidixic acid, norfloxacin,ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin),tetracyclines (e.g., doxycycline, minocycline, tetracycline), andglycopeptides (e.g., vancomycin, teicoplanin), for example. Otherantibiotics include chloramphenicol, clindamycin, trimethoprim,sulfamethoxazole, nitrofurantoin, rifampin, linezolid, synercid,polymyxin B, colisitin, colimycin, methotrexate, daptomycin,phosphonomycin and mupirocin.

The peptides and/or analogs or derivatives thereof may be administeredto any host, including a human or non-human animal, in an amounteffective to inhibit not only growth of a bacterium, but also a virus,parasite or fungus. These peptides are useful as antimicrobial agents,antiviral agents, and antifungal agents. The peptides and/or analogs orderivatives thereof may be administered to any host, including a humanor non-human animal, in an amount effective to inhibit not only growthof a bacterium, but also a virus or fungus. These peptides are useful asantimicrobial agents, antiviral agents, and antifungal agents.

In addition to being active against a broad range of pathogens,bactenecin has been shown to be cytotoxic to rat embryonic neurons,fetal rat astrocytes and human glioblastoma cells (Radermacher et al.,J. Neuro. Res. 36:657, 1993). Thus, it is envisioned that the peptidesof the present invention can be used to inhibit the growth of aeukaryotic cell by contacting the eukaryotic cell with an inhibitingeffective amount of a peptide of the invention. Such a method would beuseful, for example, for inhibiting a cell proliferation-associateddisorder in a subject having or at risk of having such a disorder. Themethod can involve, for example, administering to the subject atherapeutically effective amount of a peptide of the present inventionto inhibit the over-growth of cells in a subject in need of suchtreatment. Such disorders would include, for example, neurologicalrelated disorders.

The invention further provides a method of protecting objects frombacterial colonization. The peptides of the invention remain active whenconjugated to solid surfaces. Thus, the peptides may be used forprotecting objects such as medical devices from colonization withpathogenic bacteria by chemically conjugating, or coating by any othermeans, at least one peptide of the invention to the surface of themedical device. Such medical devices include indwelling catheters, andthe like.

I. Methods of Use—Immunomodulatory

The present invention provides novel cationic peptides, characterized bya group of generic formulas which have ability to modulate (e.g., up-and/or down regulate) polypeptide expression, thereby regulating sepsisand inflammatory responses and/or innate immunity.

“Innate immunity” as used herein refers to the natural ability of anorganism to defend itself against invasions by pathogens. Pathogens ormicrobes as used herein, may include, but are not limited to bacteria,fungi, parasite, and viruses. Innate immunity is contrasted withacquired/adaptive immunity in which the organism develops a defensivemechanism based substantially on antibodies and/or immune lymphocytesthat is characterized by specificity, amplifiability and self vs.non-self discrimination. With innate immunity, broad, nonspecificimmunity is provided and there is no immunologic memory of priorexposure. The hallmarks of innate immunity are effectiveness against abroad variety of potential pathogens, independence of prior exposure toa pathogen, and immediate effectiveness (in contrast to the specificimmune response which takes days to weeks to be elicited). In addition,innate immunity includes immune responses that affect other diseases,such as cancer, inflammatory diseases, multiple sclerosis, various viralinfections, and the like.

In innate immunity, the immune response is not dependent upon antigens.The innate immunity process may include the production of secretorymolecules and cellular components as set forth above. In innateimmunity, the pathogens are recognized by receptors (for example,Toll-like receptors) that have broad specificity, are capable ofrecognizing many pathogens, and are encoded in the germline. TheseToll-like receptors have broad specificity and are capable ofrecognizing many pathogens. When cationic peptides are present in theimmune response, they aid in the host response to pathogens. This changein the immune response induces the release of chemokines, which promotethe recruitment of immune cells to the site of infection.

Chemokines, or chemoattractant cytokines, are a subgroup of immunefactors that mediate chemotactic and other pro-inflammatory phenomena(See, Schall, 1991, Cytokine 3:165-183). Chemokines are small moleculesof approximately 70-80 residues in length and can generally be dividedinto two subgroups, a which have two N-terminal cysteines separated by asingle amino acid (C×C) and β which have two adjacent cysteines at the Nterminus (CC). RANTES, MIP-1α and MIP-1β are members of the β subgroup(reviewed by Horuk, R., 1994, Trends Pharmacol. Sci, 15:159-165; Murphy,P. M., 1994, Annu. Rev. Immunol., 12:593-633). The amino terminus of theβ chemokines RANTES, MCP-1, and MCP-3 have been implicated in themediation of cell migration and inflammation induced by thesechemokines. This involvement is suggested by the observation that thedeletion of the amino terminal 8 residues of MCP-1, amino terminal 9residues of MCP-3, and amino terminal 8 residues of RANTES and theaddition of a methionine to the amino terminus of RANTES, antagonize thechemotaxis, calcium mobilization and/or enzyme release stimulated bytheir native counterparts (Gong et al., 1996 J. Biol. Chem.271:10521-10527; Proudfoot et al., 1996 J. Biol. Chem. 271:2599-2603).Additionally, a chemokine-like chemotactic activity has been introducedinto MCP-1 via a double mutation of Tyr 28 and Arg 30 to leucine andvaline, respectively, indicating that internal regions of this proteinalso play a role in regulating chemotactic activity (Beall et al., 1992,J. Biol. Chem. 267:3455-3459).

The monomeric forms of all chemokines characterized thus far sharesignificant structural homology, although the quaternary structures of αand β groups are distinct. While the monomeric structures of the β and αchemokines are very similar, the dimeric structures of the two groupsare completely different. An additional chemokine, lymphotactin, whichhas only one N-terminal cysteine has also been identified and mayrepresent an additional subgroup (γ) of chemokines (Yoshida et al.,1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science266:1395-1399).

Receptors for chemokines belong to the large family of G-proteincoupled, 7 transmembrane domain receptors (GCR's) (See, reviews byHoruk, R., 1994, Trends Pharmacol. Sci. 15:159-165; and Murphy, P. M.,1994, Annu. Rev. Immunol. 12:593-633). Competition binding andcross-desensitization studies have shown that chemokine receptorsexhibit considerable promiscuity in ligand binding. Examplesdemonstrating the promiscuity among β chemokine receptors include: CCCKR-1, which binds RANTES and MIP-1α (Neote et al., 1993, Cell 72:415-425), CC CKR-4, which binds RANTES, MIP-1α, and MCP-1 (Power et al.,1995, J. Biol. Chem. 270:19495-19500), and CC CKR-5, which binds RANTES,MIP-1α, and MIP-1β (Alkhatib et al., 1996, Science 272:1955-1958 andDragic et al., 1996, Nature 381:667-674). Erythrocytes possess areceptor (known as the Duffy antigen) which binds both α and βchemokines (Horuk et al., 1994, J. Biol. Chem. 269:17730-17733; Neote etal., 1994, Blood 84:44-52; and Neote et al., 1993, J. Biol. Chem.268:12247-12249). Thus the sequence and structural homologies evidentamong chemokines and their receptors allows some overlap inreceptor-ligand interactions.

In one aspect, the present invention provides the use of compoundsincluding peptides of the invention to reduce sepsis and inflammatoryresponses by acting directly on host cells. In this aspect, a method ofidentification of a polynucleotide or polynucleotides that are regulatedby one or more sepsis or inflammatory inducing agents is provided, wherethe regulation is altered by a cationic peptide. Such sepsis orinflammatory inducing agents include, but are not limited to endotoxiclipopolysaccharide (LPS), lipoteichoic acid (LTA) and/or CpG DNA orintact bacteria or other bacterial components. The identification isperformed by contacting the host cell with the sepsis or inflammatoryinducing agents and further contacting with a cationic peptide eithersimultaneously or immediately after. The expression of thepolynucleotide or polypeptide in the presence and absence of thecationic peptide is observed and a change in expression is indicative ofa polynucleotide or polypeptide or pattern of polynucleotides orpolypeptides that is regulated by a sepsis or inflammatory inducingagent and inhibited by a cationic peptide. In another aspect, theinvention provides a polynucleotide identified by the method.

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, and the like toproduce structural analogs. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, peptidiomimetics,saccharides, fatty acids, steroids, purines, pyrimidines, polypeptides,polynucleotides, chemical compounds, derivatives, structural analogs orcombinations thereof.

Generally, in the methods of the invention, a cationic peptide isutilized to detect and locate a polynucleotide or polypeptide that isessential in the process of sepsis or inflammation. Once identified, apattern of polynucleotide or polypeptide expression may be obtained byobserving the expression in the presence and absence of the cationicpeptide. The pattern obtained in the presence of the cationic peptide isthen useful in identifying additional compounds that can inhibitexpression of the polynucleotide and therefore block sepsis orinflammation. It is well known to one of skill in the art thatnon-peptidic chemicals and peptidomimetics can mimic the ability ofpeptides to bind to receptors and enzyme binding sites and thus can beused to block or stimulate biological reactions. Where an additionalcompound of interest provides a pattern of polynucleotide or polypeptideexpression similar to that of the expression in the presence of acationic peptide, that compound is also useful in the modulation ofsepsis or an innate immune response. In this manner, the cationicpeptides of the invention, which are known inhibitors of sepsis andinflammation and enhancers of innate immunity are useful as tools in theidentification of additional compounds that inhibit sepsis andinflammation and enhance innate immunity.

As can be seen in the Examples below, peptides of the invention have anability to reduce the expression of polynucleotides or polypeptidesregulated by LPS, particularly the quintessential pro-inflammatorycytokine TNFα. High levels of endotoxin in the blood are responsible formany of the symptoms seen during a serious infection or inflammationsuch as fever and an elevated white blood cell count, and many of theseeffects reflect or are caused by high levels of induced TNFα. Endotoxin(also called lipopolysaccharide) is a component of the cell wall of Gramnegative bacteria and is a potent trigger of the pathophysiology ofsepsis. The basic mechanisms of inflammation and sepsis are related.

In another aspect, the invention identifies agents that enhances innateimmunity. Human cells that contain a polynucleotide or polynucleotidesthat encode a polypeptide or polypeptides involved in innate immunityare contacted with an agent of interest. Expression of thepolynucleotide is determined, both in the presence and absence of theagent. The expression is compared and of the specific modulation ofexpression was indicative of an enhancement of innate immunity. Inanother aspect, the agent does not stimulate a septic reaction asrevealed by the lack of upregulation of the pro-inflammatory cytokineTNF-α. In still another aspect the agent reduces or blocks theinflammatory or septic response.

In another aspect, the invention provides methods of directpolynucleotide or polypeptide regulation by cationic peptides and theuse of compounds including cationic peptides to stimulate elements ofinnate immunity. In this aspect, the invention provides a method ofidentification of a pattern of polynucleotide or polypeptide expressionfor identification of a compound that enhances innate immunity. In themethod of the invention, an initial detection of a pattern ofpolypeptide expression for cells contacted in the presence and absenceof a cationic peptide is made. The pattern resulting from polypeptideexpression in the presence of the peptide represents stimulation ofinnate immunity. A pattern of polypeptide expression is then detected inthe presence of a test compound, where a resulting pattern with the testcompound that is similar to the pattern observed in the presence of thecationic peptide is indicative of a compound that enhances innateimmunity. In another aspect, the invention provides compounds that areidentified in the above methods. In another aspect, the compound of theinvention stimulates chemokine expression. Chemokine or chemokinereceptors may include, but are not limited to IL8, Gro-α, MCP-1, andMCP-3. In still another aspect, the compound is a peptide,peptidomimetic, chemical compound, or a nucleic acid molecule.

It is shown below, for example, in FIG. 2, that cationic peptides canneutralize the host response to the signaling molecules of infectiousagents as well as modify the transcriptional responses of host cells,mainly by down-regulating the pro-inflammatory response and/orup-regulating the anti-inflammatory response. Example 5 shows that thecationic peptides can selectively suppress the induction of the sepsisinducing cytokine TNFα in host cells. Example 6 shows that the cationicpeptides can aid in the host response to pathogens by inducing therelease of chemokines, which promote the recruitment of immune cells tothe site of infection.

It is seen from the examples below that cationic peptides have asubstantial influence on the host response to pathogens in that theyassist in regulation of the host immune response by inducing selectivepro-inflammatory responses that for example promote the recruitment ofimmune cells to the site of infection but not inducing potentiallyharmful pro-inflammatory cytokines. Sepsis appears to be caused in partby an overwhelming pro-inflammatory response to infectious agents.Peptides can aid the host in a “balanced” response to pathogens byinducing an anti-inflammatory response and suppressing certainpotentially harmful pro-inflammatory responses.

The present invention features methods for enhancing a vaccine-inducedadaptive immune response in a subject comprising administering to thesubject an adjuvant composition comprising a pharmaceutically acceptablecarrier and an immunomodulatory peptides of the invention in an amounteffective to enhance the vaccine-induced adaptive immune response in thesubject. In some aspects, the methods comprise administering to asubject an effective amount of an adjuvant composition comprising apharmaceutically effective carrier and a polypeptide having the aminoacid sequence SEQ ID NO: 2, 7, 12, 15, 1213, 1214, 1215, 1216, 1221,1222, 1223, 1224, 1229, 1230, 1231, 1232, 1237, 1238, 1239, 1240, 1245,1246, 1248, or analogs, derivatives, amidated variations andconservative variations thereof. In other aspects, the methods compriseadministering to a subject an effective amount of an adjuvantcomposition comprising a pharmaceutically effective carrier and apolypeptide having the amino acid sequence SEQ ID NO: 1020, 1021, 1022,1032, 1065, 1069, 1078, 1081, 1087, 1089, 1135, 1145, 1160, 1217, 1218,1219, 1220, 1225, 1227, 1228, 1233, 1234, 1241, 1242, 1243, 1244, 1250,1251, 1252, or analogs, derivatives, amidated variations andconservative variations thereof. In other aspects, the methods compriseadministering to a subject an effective amount of an adjuvantcomposition comprising a pharmaceutically effective carrier and apolypeptide having the amino acid sequence SEQ ID NO: 18, 1253, 1255,1256, 1257, 1258, or analogs, derivatives, amidated variations andconservative variations thereof.

The vaccine compositions can comprise agents that enhance the protectiveefficacy of the vaccine, such as adjuvants. Adjuvants include anycompound or compounds that acts to enhance a vaccine-induced adaptiveimmune response, thereby reducing the quantity of antigen necessary inthe vaccine, and/or the frequency of administration necessary togenerate a protective immune response. Adjuvants can include forexample, emulsifiers, muramyl dipeptides, pyridine, aqueous adjuvantssuch as aluminum hydroxide, chitosan-based adjuvants, and any of thevarious saponins, oils, and other substances known in the art, such asAmphigen, LPS, bacterial cell wall extracts, bacterial DNA, syntheticoligonucleotides and combinations thereof (Schijns et al. (2000) Curr.Opin. Immunol. 12:456), Mycobacterialphlei (M. phlei) cell wall extract(MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA (M-DNA), M-DNA-M. phleicell wall complex (MCC). Compounds which can serve as emulsifiersinclude natural and synthetic emulsifying agents, as well as anionic,cationic and nonionic compounds. Among the synthetic compounds, anionicemulsifying agents include, for example, the potassium, sodium andammonium salts of lauric and oleic acid, the calcium, magnesium andaluminum salts of fatty acids, and organic sulfonates such as sodiumlauryl sulfate. Synthetic cationic agents include, for example,cetyltrhethylammonlum bromide, while synthetic nonionic agents areexemplified by glycerylesters (e.g., glyceryl monostearate),polyoxyethylene glycol esters and ethers, and the sorbitan fatty acidesters (e.g., sorbitan monopalmitate) and their polyoxyethylenederivatives (e.g., polyoxyethylene sorbitan monopalmitate). Naturalemulsifying agents include acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as asingle oil, a mixture of oils, a water-in-oil emulsion, or anoil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, oran animal oil. Mineral oils are liquid hydrocarbons obtained frompetrolatum via a distillation technique, and are also referred to in theart as liquid paraffin, liquid petrolatum, or white mineral oil.Suitable animal oils include, for example, cod liver oil, halibut oil,menhaden oil, orange roughy oil and shark liver oil, all of which areavailable commercially. Suitable vegetable oils, include, for example,canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil,safflower oil, sesame oil, soybean oil, and the like. Freund's CompleteAdjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) are two commonadjuvants that are commonly used in vaccine preparations, and are alsosuitable for use in the present invention. Both FCA and FIA arewater-in-mineral oil emuslsions; however, FCA also contains a killedMycobacterium sp.

Immunomodulatory cytokines can also be used in the vaccine compositionsto enhance vaccine efficacy, for example, as an adjuvant. Non-limitingexamples of such cytokines include interferon alpha (IFN-α),interleukin-2 (IL-2), and granulocyte macrophage-colony stimulatingfactor (GM-CSF), or combinations thereof. GM-CSF is highly preferred.

Vaccine compositions comprising the immunomodulatory peptides of theinvention and further comprising adjuvants can be prepared usingtechniques well known to those skilled in the art including, but notlimited to, mixing, sonication and microfluidation. The adjuvant cancomprise from about 10% to about 50% (v/v) of the vaccine composition,more preferably about 20% to about 40% (v/v), and more preferably about20% to about 30% (v/v), or any integer within these ranges. About 25%(v/v) is highly preferred.

J. Treatment Regimes

The invention provides pharmaceutical compositions comprising one or acombination of antimicrobial peptides, for example, formulated togetherwith a pharmaceutically acceptable carrier. Some compositions include acombination of multiple (e.g., two or more) peptides of the invention.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one aspect, the carrier is suitable forparenteral administration. Alternatively, the carrier can be suitablefor intravenous, intraperitoneal or intramuscular administration. Inanother aspect, the carrier is suitable for oral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is compatible with theactive compound, use thereof in the pharmaceutical compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (See, e.g., Berge, et al., J. Pharm.Sci., 66: 1-19, 1977). Examples of such salts include acid additionsalts and base addition salts. Acid addition salts include those derivedfrom nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well asfrom nontoxic organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,aromatic acids, aliphatic and aromatic sulfonic acids and the like. Baseaddition salts include those derived from alkaline earth metals, such assodium, potassium, magnesium, calcium and the like, as well as fromnontoxic organic amines, such as N,N′-dibenzylethylenediamine,N-methylglucamine, chloroprocaine, choline, diethanolamine,ethylenediamine, procaine and the like.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of adisease or condition (i.e., as a result of bacteria, fungi, viruses,parasites or the like) in an amount sufficient to eliminate or reducethe risk, lessen the severity, or delay the outset of the disease,including biochemical, histologic and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. In therapeuticapplications, compositions or medicants are administered to a patientsuspected of, or already suffering from such a disease or condition inan amount sufficient to cure, or at least partially arrest, the symptomsof the disease or condition (e.g., biochemical and/or histologic),including its complications and intermediate pathological phenotypes indevelopment of the disease or condition. An amount adequate toaccomplish therapeutic or prophylactic treatment is defined as atherapeutically- or prophylactically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient response has been achieved.Typically, the response is monitored and repeated dosages are given ifthe response starts to wane.

The pharmaceutical composition of the present invention should besterile and fluid to the extent that the composition is deliverable bysyringe. In addition to water, the carrier can be an isotonic bufferedsaline solution, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. Proper fluidity can be maintained, for example, by useof coating such as lecithin, by maintenance of required particle size inthe case of dispersion and by use of surfactants. In many cases, it ispreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Long-term absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound can be orally administered, for example, with an inert diluentor an assimilable edible carrier.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, intreatment of bacteria, the combination therapy can include a compositionof the present invention with at least one agent or other conventionaltherapy.

K. Routes of Administration

A composition of the present invention can be administered by a varietyof methods known in the art. The route and/or mode of administrationvary depending upon the desired results. The phrases “parenteraladministration” and “administered parenterally” mean modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. The peptide of the invention can beadministered parenterally by injection or by gradual infusion over time.The peptide can also be prepared with carriers that protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Further methods for delivery of the peptide include orally, byencapsulation in microspheres or proteinoids, by aerosol delivery to thelungs, or transdermally by iontophoresis or transdermal electroporation.To administer a peptide of the invention by certain routes ofadministration, it can be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.The method of the invention also includes delivery systems such asmicroencapsulation of peptides into liposomes or a diluent.Microencapsulation also allows co-entrapment of antimicrobial moleculesalong with the antigens, so that these molecules, such as antibiotics,may be delivered to a site in need of such treatment in conjunction withthe peptides of the invention. Liposomes in the blood stream aregenerally taken up by the liver and spleen. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., J. Neuroimmunol., 7: 27, 1984). Thus, the method of theinvention is particularly useful for delivering antimicrobial peptidesto such organs. Biodegradable, biocompatible polymers can be used, suchas ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are described by e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, Ed., 1978, Marcel Dekker,Inc., New York. Other methods of administration will be known to thoseskilled in the art.

Preparations for parenteral administration of a peptide of the inventioninclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

Therapeutic compositions typically must be sterile, substantiallyisotonic, and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it is preferable to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. Therapeutic compositionscan also be administered with medical devices known in the art. Forexample, in a preferred aspect, a therapeutic composition of theinvention can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in, e.g., U.S. Pat. No. 5,399,163,5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.Examples of implants and modules useful in the present inventioninclude: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which discloses a therapeutic device foradministering medicants through the skin; U.S. Pat. No. 4,447,233, whichdiscloses a medication infusion pump for delivering medication at aprecise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known.

When the peptides of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (or0.1 to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

L. Effective Dosages

“Therapeutically effective amount” as used herein for treatment ofantimicrobial related diseases and conditions refers to the amount ofpeptide used that is of sufficient quantity to decrease the numbers ofbacteria, viruses, fungi, and parasites in the body of a subject. Thedosage ranges for the administration of peptides are those large enoughto produce the desired effect. The amount of peptide adequate toaccomplish this is defined as a “therapeutically effective dose.” Thedosage schedule and amounts effective for this use, i.e., the “dosingregimen,” will depend upon a variety of factors, including the stage ofthe disease or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age, pharmaceutical formulation and concentration of active agent, andthe like. In calculating the dosage regimen for a patient, the mode ofadministration also is taken into consideration. The dosage regimen mustalso take into consideration the pharmacokinetics, i.e., thepharmaceutical composition's rate of absorption, bioavailability,metabolism, clearance, and the like. See, e.g., the latest Remington's(Remington's Pharmaceutical Science, Mack Publishing Company, Easton,Pa.); Egleton, Peptides 18: 1431-1439, 1997; Langer Science 249:1527-1533, 1990. The dosage regimen can be adjusted by the individualphysician in the event of any contraindications.

Dosage regimens of the pharmaceutical compositions of the presentinvention are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus can be administered,several divided doses can be administered over time or the dose can beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Administration of the vaccine compositions can be by infusion orinjection (e.g., intravenously, intramuscularly, intracutaneously,subcutaneously, intrathecal, intraduodenally, intraperitoneally, and thelike). The vaccine compositions can also be administered intranasally,vaginally, rectally, orally, or transdermally as discussed herein.Preferably, the compositions are administered by intradermal injection.Administration can be at the direction of a physician.

The injections can be split into multiple injections, with such splitinoculations administered preferably substantially concurrently. Whenadministered as a split inoculation, the dose of the immunogen ispreferably, but not necessarily, proportioned equally in each separateinjection. If an adjuvant is present in the vaccine composition, thedose of the adjuvant is preferably, but not necessarily, proportionedequally in each separate injection. The separate injections for thesplit inoculation are preferably administered substantially proximal toeach other on the patient's body. In some preferred aspects, theinjections are administered at least about 1 cm apart from each other onthe body. In some preferred aspects, the injections are administered atleast about 2.5 cm apart from each other on the body. In highlypreferred aspects, the injections are administered at least about 5 cmapart from each other on the body. In some aspects, the injections areadministered at least about 10 cm apart from each other on the body. Insome aspects, the injections are administered more than 10 cm apart fromeach other on the body, for example, at least about 12.5. 15, 17.5, 20,or more cm apart from each other on the body. Primary immunizationinjections and booster injections can be administered as a splitinoculation as described and exemplified herein.

In some aspects, patients can be administered the vaccine compositions1, 2, 3, 4, 5, 6, 7, 8, or more times per month. Four to six times permonth are preferred to establish the protective immune response,particularly with respect to the primary immunization schedule. In someaspects, boosters can be administered at regular intervals such as every2, 3, 4, 5, or 6 days, every 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks,or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.Administration of the booster is preferably every 6 months. Boosters canalso be administered on an as-needed basis.

The vaccine administration schedule, including primary immunization andbooster administration, can continue as long as needed for the patient,for example, over the course of several weeks, to several months, toseveral years, to over the lifetime of the patient. In some aspects, thevaccine schedule includes more frequent administration at the beginningof the vaccine regimen, and includes less frequent administration (e.g.,boosters) over time to maintain the protective immunity. “Booster”refers to a dose of an immunogen administered to a patient to enhance,prolong, or maintain protective immunity.

The vaccines can be administered at lower doses at the beginning of thevaccine regimen, with higher doses administered over time. The vaccinescan also be administered at higher doses at the beginning of the vaccineregimen, with lower doses administered over time. The frequency ofprimary vaccine and booster administration and dose of theimmunomodulatory peptides of the invention administered can be tailoredand/or adjusted to meet the particular needs of individual patients, asdetermined by the administering physician according to any meanssuitable in the art.

In some aspects, the vaccine compositions, including compositions foradministration as a booster, comprise from about 0.001 mg to about 10 mgof the immunomodulatory peptide or peptides. In some preferred aspects,the compositions comprise about 0.1 mg of the immunomodulatory peptideor peptides. In some preferred aspects, the compositions comprise about0.5 mg of the immunomodulatory peptide or peptides. In some preferredaspects, the compositions comprise about 1 mg of the immunomodulatorypeptide or peptides.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the compounds of theinvention employed in the pharmaceutical composition at levels lowerthan that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, a suitable daily dose of a compound of the invention is thatamount of the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose generally depends upon thefactors described above. It is preferred that administration beintravenous, intramuscular, intraperitoneal, or subcutaneous, oradministered proximal to the site of the target. If desired, theeffective daily dose of a therapeutic composition can be administered astwo, three, four, five, six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

An effective dose of each of the peptides disclosed herein as potentialtherapeutics for use in treating microbial diseases and conditions isfrom about 1 μg to 500 mg/kg body weight, per single administration,which can readily be determined by one skilled in the art. As discussedabove, the dosage depends upon the age, sex, health, and weight of therecipient, kind of concurrent therapy, if any, and frequency oftreatment. Other effective dosage range upper limits are 100 mg/kg bodyweight, 50 mg/kg body weight, 25 mg/kg body weight, and 10 mg/kg bodyweight.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patent can beadministered a prophylactic regime.

Some compounds of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention cross the BBB (if desired), theycan be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, See, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes can comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (See, e.g., Ranade, J. Clin. Pharmacol., 29: 685,1989). Exemplary targeting moieties include folate or biotin (See, e.g.,U.S. Pat. No. 5,416,016 to Low, et al.); mannosides (Umezawa, et al.,Biochem. Biophys. Res. Commun., 153: 1038, 1988); antibodies (Bloeman,et al., FEBS Lett., 357: 140, 1995; Owais, et al., Antimicrob. AgentsChemother., 39: 180, 1995); surfactant protein A receptor (Briscoe, etal., Am. J. Physiol., 1233: 134, 1995), different species of which cancomprise the formulations of the inventions, as well as components ofthe invented molecules; p120 (Schreier, et al., J. Biol. Chem., 269:9090, 1994). See also Keinanen, et al., FEBS Lett., 346: 123, 1994;Killion, et al., Immunomethods, 4: 273, 1994. In some methods, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred aspect, the liposomes include a targeting moiety. In somemethods, the therapeutic compounds in the liposomes are delivered bybolus injection to a site proximal to the tumor or infection. Thecomposition should be fluid to the extent that easy syringabilityexists. It should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi.

“Bactericidal amount” as used herein refers to an amount sufficient toachieve a bacteria-killing blood concentration in the subject receivingthe treatment. The bactericidal amount of antibiotic generallyrecognized as safe for administration to a human is well known in theart, and as is known in the art, varies with the specific antibiotic andthe type of bacterial infection being treated.

Because of the antibiotic, antimicrobial, and antiviral properties ofthe peptides, they may also be used as preservatives or sterillants ofmaterials susceptible to microbial or viral contamination. The peptidesof the invention can be utilized as broad spectrum antimicrobial agentsdirected toward various specific applications. Such applications includeuse of the peptides as preservatives in processed foods (organismsincluding Salmonella, Yersinia, and Shigella), either alone or incombination with antibacterial food additives such as lysozymes; as atopical agent (Pseudomonas, Streptococcus) and to kill odor producingmicrobes (Micrococci). The relative effectiveness of the peptides of theinvention for the applications described can be readily determined byone of skill in the art by determining the sensitivity of any organismto one of the peptides.

M. Formulation

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998.Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24,1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

From the foregoing description, various modifications and changes in thecompositions and methods will occur to those skilled in the art. Allsuch modifications coming within the scope of the appended claims areintended to be included therein. Each recited range includes allcombinations and sub-combinations of ranges, as well as specificnumerals contained therein.

All publications and patent documents cited above are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and can be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

EXEMPLARY EMBODIMENTS Example 1

Materials, Methods and Peptides

Peptide Synthesis—Peptide syntheses on cellulose were performed using apipetting robot (Abimed, Langenfeld, Germany) and Whatman 50 cellulosemembranes (Whatman, Maidstone, United Kingdom) as described previously(Kramer A, Schuster A, Reinecke U, Malin R, Volkmer-Engert R, LandgrafC, Schneider-Mergener J. 1994. Combinatorial cellulose-bound peptidelibraries: screening tool for the identification of peptides that bindligands with predefined specificity. Comp. Meth. Enzymol. 6, 388-395;Kramer A, Keitel T, Winkler K, Stocklein W, Hohne W, Schneider-MergenerJ. 1997. Molecular basis for the binding promiscuity of an anti-p24(HIV-1) monoclonal antibody. Cell 91, 799-809). The HPLC purifiedpeptides used for further characterization (CD, membrane permeability,MIC) were purchased from Thermo electron cooperation (Ulm, Germany).Table 2A

TABLE 2A Peptides utilized in these studies. All peptides were amidatedat the C-terminus unless otherwise noted. Name Sequence ID HH1QRLRIRVAVIRA SEQ ID NO 1 HH2 VQLRIRVAVIRA SEQ ID NO 2 HH3 VRFRIRVAVIRASEQ ID NO 3 HH4 VRWRIRVAVIRA SEQ ID NO 4 HH5 VRLWIRVAVIRA SEQ ID NO 5HH6 VRLRIRVWVIRA SEQ ID NO 6 HH7 VRLRIRVAVRRA SEQ ID NO 7 HH8VRLRIRVAVIRK SEQ ID NO 8 HH9 VQLRIRVRVIRK SEQ ID NO 9 HH10 KRFRIRVAVRRASEQ ID NO 10 HH11 VRLRIRVRVIRK SEQ ID NO 11 HH12 KQFRIRVRVIRK SEQ ID NO12 HH13 HQFRFRFRVRRK SEQ ID NO 13 HH14 HQWRIRVAVRRH SEQ ID NO 14 HH15KRFRIRVRVIRK SEQ ID NO 15 HH16 KRWRIRVRVIRK SEQ ID NO 16 HH17 KIWVRWKSEQ ID NO 17 HH18 IWVIWRR SEQ ID NO 18 HH19 ALPWKWPWWPWRR SEQ ID NO 19HH20 IAPWKWPWWPWRR SEQ ID NO 20 HH21 ILAWKWPWWPWRR SEQ ID NO 21 HH22ILPAKWPWWPWRR SEQ ID NO 22 HH23 ILPWAWPWWPWRR SEQ ID NO 23 HH24ILPWKAPWWPWRR SEQ ID NO 24 HH25 ILPWKWAWWPWRR SEQ ID NO 25 HH26ILPWKWPAWPWRR SEQ ID NO 26 HH27 ILPWKWPWAPWRR SEQ ID NO 27 HH28ILPWKWPWWAWRR SEQ ID NO 28 HH29 ILPWKWPWWPARR SEQ ID NO 29 HH30ILPWKWPWWPWAR SEQ ID NO 30 HH31 ILPWKWPWWPWRA SEQ ID NO 31 HH32DLPWKWPWWPWRR SEQ ID NO 32 HH33 IDPWKWPWWPWRR SEQ ID NO 33 HH34ILDWKWPWWPWRR SEQ ID NO 34 HH35 ILPDKWPWWPWRR SEQ ID NO 35 HH36ILPWDWPWWPWRR SEQ ID NO 36 HH37 ILPWKDPWWPWRR SEQ ID NO 37 HH38ILPWKWDWWPWRR SEQ ID NO 38 HH39 ILPWKWPDWPWRR SEQ ID NO 39 HH40ILPWKWPWDPWRR SEQ ID NO 40 HH41 ILPWKWPWWDWRR SEQ ID NO 41 HH42ILPWKWPWWPDRR SEQ ID NO 42 HH43 ILPWKWPWWPWDR SEQ ID NO 43 HH44ILPWKWPWWPWRD SEQ ID NO 44 HH45 ELPWKWPWWPWRR SEQ ID NO 45 HH46IEPWKWPWWPWRR SEQ ID NO 46 HH47 ILEWKWPWWPWRR SEQ ID NO 47 HH48ILPEKWPWWPWRR SEQ ID NO 48 HH49 ILPWEWPWWPWRR SEQ ID NO 49 HH50ILPWKEPWWPWRR SEQ ID NO 50 HH51 ILPWKWEWWPWRR SEQ ID NO 51 HH52ILPWKWPEWPWRR SEQ ID NO 52 HH53 ILPWKWPWEPWRR SEQ ID NO 53 HH54ILPWKWPWWEWRR SEQ ID NO 54 HH55 ILPWKWPWWPERR SEQ ID NO 55 HH56ILPWKWPWWPWER SEQ ID NO 56 HH57 ILPWKWPWWPWRE SEQ ID NO 57 HH58FLPWKWPWWPWRR SEQ ID NO 58 HH59 IFPWKWPWWPWRR SEQ ID NO 59 HH60ILFWKWPWWPWRR SEQ ID NO 60 HH61 ILPFKWPWWPWRR SEQ ID NO 61 HH62ILPWFWPWWPWRR SEQ ID NO 62 HH63 ILPWKFPWWPWRR SEQ ID NO 63 HH64ILPWKWFWWPWRR SEQ ID NO 64 HH65 ILPWKWPFWPWRR SEQ ID NO 65 HH66ILPWKWPWFPWRR SEQ ID NO 66 HH67 ILPWKWPWWFWRR SEQ ID NO 67 HH68ILPWKWPWWPFRR SEQ ID NO 68 HH69 ILPWKWPWWPWFR SEQ ID NO 69 HH70ILPWKWPWWPWRF SEQ ID NO 70 HH71 GLPWKWPWWPWRR SEQ ID NO 71 HH72IGPWKWPWWPWRR SEQ ID NO 72 HH73 ILGWKWPWWPWRR SEQ ID NO 73 HH74ILPGKWPWWPWRR SEQ ID NO 74 HH75 ILPWGWPWWPWRR SEQ ID NO 75 HH76ILPWKGPWWPWRR SEQ ID NO 76 HH77 ILPWKWGWWPWRR SEQ ID NO 77 HH78ILPWKWPGWPWRR SEQ ID NO 78 HH79 ILPWKWPWGPWRR SEQ ID NO 79 HH80ILPWKWPWWGWRR SEQ ID NO 80 HH81 ILPWKWPWWPGRR SEQ ID NO 81 HH82ILPWKWPWWPWGR SEQ ID NO 82 HH83 ILPWKWPWWPWRG SEQ ID NO 83 HH84HLPWKWPWWPWRR SEQ ID NO 84 HH85 IHPWKWPWWPWRR SEQ ID NO 85 HH86ILHWKWPWWPWRR SEQ ID NO 86 HH87 ILPHKWPWWPWRR SEQ ID NO 87 HH88ILPWHWPWWPWRR SEQ ID NO 88 HH89 ILPWKHPWWPWRR SEQ ID NO 89 HH90ILPWKWHWWPWRR SEQ ID NO 90 HH91 ILPWKWPHWPWRR SEQ ID NO 91 HH92ILPWKWPWHPWRR SEQ ID NO 92 HH93 ILPWKWPWWHWRR SEQ ID NO 93 HH94ILPWKWPWWPHRR SEQ ID NO 94 HH95 ILPWKWPWWPWHR SEQ ID NO 95 HH96ILPWKWPWWPWRH SEQ ID NO 96 HH97 IIPWKWPWWPWRR SEQ ID NO 97 HH98ILIWKWPWWPWRR SEQ ID NO 98 HH99 ILPIKWPWWPWRR SEQ ID NO 99 HH100ILPWIWPWWPWRR SEQ ID NO 100 HH101 ILPWKIPWWPWRR SEQ ID NO 101 HH102ILPWKWIWWPWRR SEQ ID NO 102 HH103 ILPWKWPIWPWRR SEQ ID NO 103 HH104ILPWKWPWIPWRR SEQ ID NO 104 HH105 ILPWKWPWWIWRR SEQ ID NO 105 HH106ILPWKWPWWPIRR SEQ ID NO 106 HH107 ILPWKWPWWPWIR SEQ ID NO 107 HH108ILPWKWPWWPWRI SEQ ID NO 108 HH109 KLPWKWPWWPWRR SEQ ID NO 109 HH110IKPWKWPWWPWRR SEQ ID NO 110 HH111 ILKWKWPWWPWRR SEQ ID NO 111 HH112ILPKKWPWWPWRR SEQ ID NO 112 HH113 ILPWKKPWWPWRR SEQ ID NO 113 HH114ILPWKWKWWPWRR SEQ ID NO 114 HH115 ILPWKWPKWPWRR SEQ ID NO 115 HH116ILPWKWPWKPWRR SEQ ID NO 116 HH117 ILPWKWPWWKWRR SEQ ID NO 117 HH118ILPWKWPWWPKRR SEQ ID NO 118 HH119 ILPWKWPWWPWKR SEQ ID NO 119 HH120ILPWKWPWWPWRK SEQ ID NO 120 HH121 LLPWKWPWWPWRR SEQ ID NO 121 HH122ILLWKWPWWPWRR SEQ ID NO 122 HH123 ILPLKWPWWPWRR SEQ ID NO 123 HH124ILPWLWPWWPWRR SEQ ID NO 124 HH125 ILPWKLPWWPWRR SEQ ID NO 125 HH126ILPWKWLWWPWRR SEQ ID NO 126 HH127 ILPWKWPLWPWRR SEQ ID NO 127 HH128ILPWKWPWLPWRR SEQ ID NO 128 HH129 ILPWKWPWWLWRR SEQ ID NO 129 HH130ILPWKWPWWPLRR SEQ ID NO 130 HH131 ILPWKWPWWPWLR SEQ ID NO 131 HH132ILPWKWPWWPWRL SEQ ID NO 132 HH133 MLPWKWPWWPWRR SEQ ID NO 133 HH134IMPWKWPWWPWRR SEQ ID NO 134 HH135 ILMWKWPWWPWRR SEQ ID NO 135 HH136ILPMKWPWWPWRR SEQ ID NO 136 HH137 ILPWMWPWWPWRR SEQ ID NO 137 HH138ILPWKMPWWPWRR SEQ ID NO 138 HH139 ILPWKWMWWPWRR SEQ ID NO 139 HH140ILPWKWPMWPWRR SEQ ID NO 140 HH141 ILPWKWPWMPWRR SEQ ID NO 141 HH142ILPWKWPWWMWRR SEQ ID NO 142 HH143 ILPWKWPWWPMRR SEQ ID NO 143 HH144ILPWKWPWWPWMR SEQ ID NO 144 HH145 ILPWKWPWWPWRM SEQ ID NO 145 HH146NLPWKWPWWPWRR SEQ ID NO 146 HH147 INPWKWPWWPWRR SEQ ID NO 147 HH148ILNWKWPWWPWRR SEQ ID NO 148 HH149 ILPNKWPWWPWRR SEQ ID NO 149 HH150ILPWNWPWWPWRR SEQ ID NO 150 HH151 ILPWKNPWWPWRR SEQ ID NO 151 HH152ILPWKWNWWPWRR SEQ ID NO 152 HH153 ILPWKWPNWPWRR SEQ ID NO 153 HH154ILPWKWPWNPWRR SEQ ID NO 154 HH155 ILPWKWPWWNWRR SEQ ID NO 155 HH156ILPWKWPWWPNRR SEQ ID NO 156 HH157 ILPWKWPWWPWNR SEQ ID NO 157 HH158ILPWKWPWWPWRN SEQ ID NO 158 HH159 PLPWKWPWWPWRR SEQ ID NO 159 HH160IPPWKWPWWPWRR SEQ ID NO 160 HH161 ILPPKWPWWPWRR SEQ ID NO 161 HH162ILPWPWPWWPWRR SEQ ID NO 162 HH163 ILPWKPPWWPWRR SEQ ID NO 163 HH164ILPWKWPPWPWRR SEQ ID NO 164 HH165 ILPWKWPWPPWRR SEQ ID NO 165 HH166ILPWKWPWWPPRR SEQ ID NO 166 HH167 ILPWKWPWWPWPR SEQ ID NO 167 HH168ILPWKWPWWPWRP SEQ ID NO 168 HH169 QLPWKWPWWPWRR SEQ ID NO 169 HH170IQPWKWPWWPWRR SEQ ID NO 170 HH171 ILQWKWPWWPWRR SEQ ID NO 171 HH172ILPQKWPWWPWRR SEQ ID NO 172 HH173 ILPWQWPWWPWRR SEQ ID NO 173 HH174ILPWKQPWWPWRR SEQ ID NO 174 HH175 ILPWKWQWWPWRR SEQ ID NO 175 HH176ILPWKWPQWPWRR SEQ ID NO 176 HH177 ILPWKWPWQPWRR SEQ ID NO 177 HH178ILPWKWPWWQWRR SEQ ID NO 178 HH179 ILPWKWPWWPQRR SEQ ID NO 179 HH180ILPWKWPWWPWQR SEQ ID NO 180 HH181 ILPWKWPWWPWRQ SEQ ID NO 181 HH182RLPWKWPWWPWRR SEQ ID NO 182 HH183 IRPWKWPWWPWRR SEQ ID NO 183 HH184ILRWKWPWWPWRR SEQ ID NO 184 HH185 ILPRKWPWWPWRR SEQ ID NO 185 HH186ILPWRWPWWPWRR SEQ ID NO 186 HH187 ILPWKRPWWPWRR SEQ ID NO 187 HH188ILPWKWRWWPWRR SEQ ID NO 188 HH189 ILPWKWPRWPWRR SEQ ID NO 189 HH190ILPWKWPWRPWRR SEQ ID NO 190 HH191 ILPWKWPWWRWRR SEQ ID NO 191 HH192ILPWKWPWWPRRR SEQ ID NO 192 HH193 SLPWKWPWWPWRR SEQ ID NO 193 HH194ISPWKWPWWPWRR SEQ ID NO 194 HH195 ILSWKWPWWPWRR SEQ ID NO 195 HH196ILPSKWPWWPWRR SEQ ID NO 196 HH197 ILPWSWPWWPWRR SEQ ID NO 197 HH198ILPWKSPWWPWRR SEQ ID NO 198 HH199 ILPWKWSWWPWRR SEQ ID NO 199 HH200ILPWKWPSWPWRR SEQ ID NO 200 HH201 ILPWKWPWSPWRR SEQ ID NO 201 HH202ILPWKWPWWSWRR SEQ ID NO 202 HH203 ILPWKWPWWPSRR SEQ ID NO 203 HH204ILPWKWPWWPWSR SEQ ID NO 204 HH205 ILPWKWPWWPWRS SEQ ID NO 205 HH206TLPWKWPWWPWRR SEQ ID NO 206 HH207 ITPWKWPWWPWRR SEQ ID NO 207 HH208ILTWKWPWWPWRR SEQ ID NO 208 HH209 ILPTKWPWWPWRR SEQ ID NO 209 HH210ILPWTWPWWPWRR SEQ ID NO 210 HH211 ILPWKTPWWPWRR SEQ ID NO 211 HH212ILPWKWTWWPWRR SEQ ID NO 212 HH213 ILPWKWPTWPWRR SEQ ID NO 213 HH214ILPWKWPWTPWRR SEQ ID NO 214 HH215 ILPWKWPWWTWRR SEQ ID NO 215 HH216ILPWKWPWWPTRR SEQ ID NO 216 HH217 ILPWKWPWWPWTR SEQ ID NO 217 HH218ILPWKWPWWPWRT SEQ ID NO 218 HH219 VLPWKWPWWPWRR SEQ ID NO 219 HH220IVPWKWPWWPWRR SEQ ID NO 220 HH221 ILVWKWPWWPWRR SEQ ID NO 221 HH222ILPVKWPWWPWRR SEQ ID NO 222 HH223 ILPWVWPWWPWRR SEQ ID NO 223 HH224ILPWKVPWWPWRR SEQ ID NO 224 HH225 ILPWKWVWWPWRR SEQ ID NO 225 HH226ILPWKWPVWPWRR SEQ ID NO 226 HH227 ILPWKWPWVPWRR SEQ ID NO 227 HH228ILPWKWPWWVWRR SEQ ID NO 228 HH229 ILPWKWPWWPVRR SEQ ID NO 229 HH230ILPWKWPWWPWVR SEQ ID NO 230 HH231 ILPWKWPWWPWRV SEQ ID NO 231 HH232WLPWKWPWWPWRR SEQ ID NO 232 HH233 IWPWKWPWWPWRR SEQ ID NO 233 HH234ILWWKWPWWPWRR SEQ ID NO 234 HH235 ILPWWWPWWPWRR SEQ ID NO 235 HH236ILPWKWWWWPWRR SEQ ID NO 236 HH237 ILPWKWPWWWWRR SEQ ID NO 237 HH238ILPWKWPWWPWWR SEQ ID NO 238 HH239 ILPWKWPWWPWRW SEQ ID NO 239 HH240YLPWKWPWWPWRR SEQ ID NO 240 HH241 IYPWKWPWWPWRR SEQ ID NO 241 HH242ILYWKWPWWPWRR SEQ ID NO 242 HH243 ILPYKWPWWPWRR SEQ ID NO 243 HH244ILPWYWPWWPWRR SEQ ID NO 244 HH245 ILPWKYPWWPWRR SEQ ID NO 245 HH246ILPWKWYWWPWRR SEQ ID NO 246 HH247 ILPWKWPYWPWRR SEQ ID NO 247 HH248ILPWKWPWYPWRR SEQ ID NO 248 HH249 ILPWKWPWWYWRR SEQ ID NO 249 HH250ILPWKWPWWPYRR SEQ ID NO 250 HH251 ILPWKWPWWPWYR SEQ ID NO 251 HH252ILPWKWPWWPWRY SEQ ID NO 252 HH253 ARLRIRVAVIRA SEQ ID NO 253 HH254DRLRIRVAVIRA SEQ ID NO 254 HH255 ERLRIRVAVIRA SEQ ID NO 255 HH256FRLRIRVAVIRA SEQ ID NO 256 HH257 GRLRIRVAVIRA SEQ ID NO 257 HH258HRLRIRVAVIRA SEQ ID NO 258 HH259 IRLRIRVAVIRA SEQ ID NO 259 HH260KRLRIRVAVIRA SEQ ID NO 260 HH261 LRLRIRVAVIRA SEQ ID NO 261 HH262MRLRIRVAVIRA SEQ ID NO 262 HH263 NRLRIRVAVIRA SEQ ID NO 263 HH264PRLRIRVAVIRA SEQ ID NO 264 HH265 QRLRIRVAVIRA SEQ ID NO 265 HH266RRLRIRVAVIRA SEQ ID NO 266 HH267 SRLRIRVAVIRA SEQ ID NO 267 HH268TRLRIRVAVIRA SEQ ID NO 268 HH269 WRLRIRVAVIRA SEQ ID NO 269 HH270YRLRIRVAVIRA SEQ ID NO 270 HH271 VALRIRVAVIRA SEQ ID NO 271 HH272VDLRIRVAVIRA SEQ ID NO 272 HH273 VELRIRVAVIRA SEQ ID NO 273 HH274VFLRIRVAVIRA SEQ ID NO 274 HH275 VGLRIRVAVIRA SEQ ID NO 275 HH276VHLRIRVAVIRA SEQ ID NO 276 HH277 VILRIRVAVIRA SEQ ID NO 277 HH278VKLRIRVAVIRA SEQ ID NO 278 HH279 VLLRIRVAVIRA SEQ ID NO 279 HH280VMLRIRVAVIRA SEQ ID NO 280 HH281 VNLRIRVAVIRA SEQ ID NO 281 HH282VPLRIRVAVIRA SEQ ID NO 282 HH283 VQLRIRVAVIRA SEQ ID NO 283 HH284VSLRIRVAVIRA SEQ ID NO 284 HH285 VTLRIRVAVIRA SEQ ID NO 285 HH286VVLRIRVAVIRA SEQ ID NO 286 HH287 VWLRIRVAVIRA SEQ ID NO 287 HH288VYLRIRVAVIRA SEQ ID NO 288 HH289 VRARIRVAVIRA SEQ ID NO 289 HH290VRDRIRVAVIRA SEQ ID NO 290 HH291 VRERIRVAVIRA SEQ ID NO 291 HH292VRFRIRVAVIRA SEQ ID NO 292 HH293 VRGRIRVAVIRA SEQ ID NO 293 HH294VRHRIRVAVIRA SEQ ID NO 294 HH295 VRIRIRVAVIRA SEQ ID NO 295 HH296VRKRIRVAVIRA SEQ ID NO 296 HH297 VRMRIRVAVIRA SEQ ID NO 297 HH298VRNRIRVAVIRA SEQ ID NO 298 HH299 VRPRIRVAVIRA SEQ ID NO 299 HH300VRQRIRVAVIRA SEQ ID NO 300 HH301 VRRRIRVAVIRA SEQ ID NO 301 HH302VRSRIRVAVIRA SEQ ID NO 302 HH303 VRTRIRVAVIRA SEQ ID NO 303 HH304VRVRIRVAVIRA SEQ ID NO 304 HH305 VRWRIRVAVIRA SEQ ID NO 305 HH306VRYRIRVAVIRA SEQ ID NO 306 HH307 VRLAIRVAVIRA SEQ ID NO 307 HH308VRLDIRVAVIRA SEQ ID NO 308 HH309 VRLEIRVAVIRA SEQ ID NO 309 HH310VRLFIRVAVIRA SEQ ID NO 310 HH311 VRLGIRVAVIRA SEQ ID NO 311 HH312VRLHIRVAVIRA SEQ ID NO 312 HH313 VRLIIRVAVIRA SEQ ID NO 313 HH314VRLKIRVAVIRA SEQ ID NO 314 HH315 VRLLIRVAVIRA SEQ ID NO 315 HH316VRLMIRVAVIRA SEQ ID NO 316 HH317 VRLNIRVAVIRA SEQ ID NO 317 HH318VRLPIRVAVIRA SEQ ID NO 318 HH319 VRLQIRVAVIRA SEQ ID NO 319 HH320VRLSIRVAVIRA SEQ ID NO 320 HH321 VRLTIRVAVIRA SEQ ID NO 321 HH322VRLVIRVAVIRA SEQ ID NO 322 HH323 VRLWIRVAVIRA SEQ ID NO 323 HH324VRLYIRVAVIRA SEQ ID NO 324 HH325 VRLRARVAVIRA SEQ ID NO 325 HH326VRLRDRVAVIRA SEQ ID NO 326 HH327 VRLRERVAVIRA SEQ ID NO 327 HH328VRLRFRVAVIRA SEQ ID NO 328 HH329 VRLRGRVAVIRA SEQ ID NO 329 HH330VRLRHRVAVIRA SEQ ID NO 330 HH331 VRLRKRVAVIRA SEQ ID NO 331 HH332VRLRLRVAVIRA SEQ ID NO 332 HH333 VRLRMRVAVIRA SEQ ID NO 333 HH334VRLRNRVAVIRA SEQ ID NO 334 HH335 VRLRPRVAVIRA SEQ ID NO 335 HH336VRLRQRVAVIRA SEQ ID NO 336 HH337 VRLRRRVAVIRA SEQ ID NO 337 HH338VRLRSRVAVIRA SEQ ID NO 338 HH339 VRLRTRVAVIRA SEQ ID NO 339 HH340VRLRVRVAVIRA SEQ ID NO 340 HH341 VRLRWRVAVIRA SEQ ID NO 341 HH342VRLRYRVAVIRA SEQ ID NO 342 HH343 VRLRIAVAVIRA SEQ ID NO 343 HH344VRLRIDVAVIRA SEQ ID NO 344 HH345 VRLRIEVAVIRA SEQ ID NO 345 HH346VRLRIFVAVIRA SEQ ID NO 346 HH347 VRLRIGVAVIRA SEQ ID NO 347 HH348VRLRIHVAVIRA SEQ ID NO 348 HH349 VRLRIIVAVIRA SEQ ID NO 349 HH350VRLRIKVAVIRA SEQ ID NO 350 HH351 VRLRILVAVIRA SEQ ID NO 351 HH352VRLRIMVAVIRA SEQ ID NO 352 HH353 VRLRINVAVIRA SEQ ID NO 353 HH354VRLRIPVAVIRA SEQ ID NO 354 HH355 VRLRIQVAVIRA SEQ ID NO 355 HH356VRLRISVAVIRA SEQ ID NO 356 HH357 VRLRITVAVIRA SEQ ID NO 357 HH358VRLRIVVAVIRA SEQ ID NO 358 HH359 VRLRIWVAVIRA SEQ ID NO 359 HH360VRLRIYVAVIRA SEQ ID NO 360 HH361 VRLRIRAAVIRA SEQ ID NO 361 HH362VRLRIRDAVIRA SEQ ID NO 362 HH363 VRLRIREAVIRA SEQ ID NO 363 HH364VRLRIRFAVIRA SEQ ID NO 364 HH365 VRLRIRGAVIRA SEQ ID NO 365 HH366VRLRIRHAVIRA SEQ ID NO 366 HH367 VRLRIRIAVIRA SEQ ID NO 367 HH368VRLRIRKAVIRA SEQ ID NO 368 HH369 VRLRIRLAVIRA SEQ ID NO 369 HH370VRLRIRMAVIRA SEQ ID NO 370 HH371 VRLRIRNAVIRA SEQ ID NO 371 HH372VRLRIRPAVIRA SEQ ID NO 372 HH373 VRLRIRQAVIRA SEQ ID NO 373 HH374VRLRIRRAVIRA SEQ ID NO 374 HH375 VRLRIRSAVIRA SEQ ID NO 375 HH376VRLRIRTAVIRA SEQ ID NO 376 HH377 VRLRIRWAVIRA SEQ ID NO 377 HH378VRLRIRYAVIRA SEQ ID NO 378 HH379 VRLRIRVDVIRA SEQ ID NO 379 HH380VRLRIRVEVIRA SEQ ID NO 380 HH381 VRLRIRVFVIRA SEQ ID NO 381 HH382VRLRIRVGVIRA SEQ ID NO 382 HH383 VRLRIRVHVIRA SEQ ID NO 383 HH384VRLRIRVIVIRA SEQ ID NO 384 HH385 VRLRIRVKVIRA SEQ ID NO 385 HH386VRLRIRVLVIRA SEQ ID NO 386 HH387 VRLRIRVMVIRA SEQ ID NO 387 HH388VRLRIRVNVIRA SEQ ID NO 388 HH389 VRLRIRVPVIRA SEQ ID NO 389 HH390VRLRIRVQVIRA SEQ ID NO 390 HH391 VRLRIRVRVIRA SEQ ID NO 391 HH392VRLRIRVSVIRA SEQ ID NO 392 HH393 VRLRIRVTVIRA SEQ ID NO 393 HH394VRLRIRVVVIRA SEQ ID NO 394 HH395 VRLRIRVWVIRA SEQ ID NO 395 HH396VRLRIRVYVIRA SEQ ID NO 396 HH397 VRLRIRVAAIRA SEQ ID NO 397 HH398VRLRIRVADIRA SEQ ID NO 398 HH399 VRLRIRVAEIRA SEQ ID NO 399 HH400VRLRIRVAFIRA SEQ ID NO 400 HH401 VRLRIRVAGIRA SEQ ID NO 401 HH402VRLRIRVAHIRA SEQ ID NO 402 HH403 VRLRIRVAIIRA SEQ ID NO 403 HH404VRLRIRVAKIRA SEQ ID NO 404 HH405 VRLRIRVALIRA SEQ ID NO 405 HH406VRLRIRVAMIRA SEQ ID NO 406 HH407 VRLRIRVANIRA SEQ ID NO 407 HH408VRLRIRVAPIRA SEQ ID NO 408 HH409 VRLRIRVAQIRA SEQ ID NO 409 HH410VRLRIRVARIRA SEQ ID NO 410 HH411 VRLRIRVASIRA SEQ ID NO 411 HH412VRLRIRVATIRA SEQ ID NO 412 HH413 VRLRIRVAWIRA SEQ ID NO 413 HH414VRLRIRVAYIRA SEQ ID NO 414 HH415 VRLRIRVAVARA SEQ ID NO 415 HH416VRLRIRVAVDRA SEQ ID NO 416 HH417 VRLRIRVAVERA SEQ ID NO 417 HH418VRLRIRVAVFRA SEQ ID NO 418 HH419 VRLRIRVAVGRA SEQ ID NO 419 HH420VRLRIRVAVHRA SEQ ID NO 420 HH421 VRLRIRVAVKRA SEQ ID NO 421 HH422VRLRIRVAVLRA SEQ ID NO 422 HH423 VRLRIRVAVMRA SEQ ID NO 423 HH424VRLRIRVAVNRA SEQ ID NO 424 HH425 VRLRIRVAVPRA SEQ ID NO 425 HH426VRLRIRVAVQRA SEQ ID NO 426 HH427 VRLRIRVAVRRA SEQ ID NO 427 HH428VRLRIRVAVSRA SEQ ID NO 428 HH429 VRLRIRVAVTRA SEQ ID NO 429 HH430VRLRIRVAVVRA SEQ ID NO 430 HH431 VRLRIRVAVWRA SEQ ID NO 431 HH432VRLRIRVAVYRA SEQ ID NO 432 HH433 VRLRIRVAVIAA SEQ ID NO 433 HH434VRLRIRVAVIDA SEQ ID NO 434 HH435 VRLRIRVAVIEA SEQ ID NO 435 HH436VRLRIRVAVIFA SEQ ID NO 436 HH437 VRLRIRVAVIGA SEQ ID NO 437 HH438VRLRIRVAVIHA SEQ ID NO 438 HH439 VRLRIRVAVIIA SEQ ID NO 439 HH440VRLRIRVAVIKA SEQ ID NO 440 HH441 VRLRIRVAVILA SEQ ID NO 441 HH442VRLRIRVAVIMA SEQ ID NO 442 HH443 VRLRIRVAVINA SEQ ID NO 443 HH444VRLRIRVAVIPA SEQ ID NO 444 HH445 VRLRIRVAVIQA SEQ ID NO 445 HH446VRLRIRVAVISA SEQ ID NO 446 HH447 VRLRIRVAVITA SEQ ID NO 447 HH448VRLRIRVAVIVA SEQ ID NO 448 HH449 VRLRIRVAVIWA SEQ ID NO 449 HH450VRLRIRVAVIYA SEQ ID NO 450 HH451 VRLRIRVAVIRD SEQ ID NO 451 HH452VRLRIRVAVIRE SEQ ID NO 452 HH453 VRLRIRVAVIRF SEQ ID NO 453 HH454VRLRIRVAVIRG SEQ ID NO 454 HH455 VRLRIRVAVIRH SEQ ID NO 455 HH456VRLRIRVAVIRI SEQ ID NO 456 HH457 VRLRIRVAVIRK SEQ ID NO 457 HH458VRLRIRVAVIRL SEQ ID NO 458 HH459 VRLRIRVAVIRM SEQ ID NO 459 HH460VRLRIRVAVIRN SEQ ID NO 460 HH461 VRLRIRVAVIRP SEQ ID NO 461 HH462VRLRIRVAVIRQ SEQ ID NO 462 HH463 VRLRIRVAVIRR SEQ ID NO 463 HH464VRLRIRVAVIRS SEQ ID NO 464 HH465 VRLRIRVAVIRT SEQ ID NO 465 HH466VRLRIRVAVIRV SEQ ID NO 466 HH467 VRLRIRVAVIRW SEQ ID NO 467 HH468VRLRIRVAVIRY SEQ ID NO 468 HH469 RRRRVKWWR SEQ ID NO 469 HH470 WLRKKQGRLSEQ ID NO 470 HH471 KWVRVYLRW SEQ ID NO 471 HH472 GKVMISIVR SEQ ID NO472 HH473 IKVVRWRWR SEQ ID NO 473 HH474 RRRRRWVRR SEQ ID NO 474 HH475HMNRFRTVY SEQ ID NO 475 HH476 VRKRGSWRM SEQ ID NO 476 HH477 RIIRTYKRGSEQ ID NO 477 HH478 WWRWRLRLI SEQ ID NO 478 HH479 WLNRLYIRL SEQ ID NO479 HH480 IWRWTKWFW SEQ ID NO 480 HH481 RFKGSWKYR SEQ ID NO 481 HH482VWVIRKKKW SEQ ID NO 482 HH483 RGRRVWRLF SEQ ID NO 483 HH484 WRWRKVKQWSEQ ID NO 484 HH485 WWKYWRKVI SEQ ID NO 485 HH486 WLVRIRKRI SEQ ID NO486 HH487 WWRWWQRRW SEQ ID NO 487 HH488 RKKWWWKIR SEQ ID NO 488 HH489WVRKKIRRR SEQ ID NO 489 HH490 RYRRRWYIR SEQ ID NO 490 HH491 LYRWVWKVGSEQ ID NO 491 HH492 VRRRWFKWL SEQ ID NO 492 HH493 RRLWWWKWL SEQ ID NO493 HH494 WRFKWTRRG SEQ ID NO 494 HH495 KWWRHRRMW SEQ ID NO 495 HH496RRKRWWWRT SEQ ID NO 496 HH497 WRRKIVRVW SEQ ID NO 497 HH498 KLRRGSLWRSEQ ID NO 498 HH499 RVIWWWRRK SEQ ID NO 499 HH500 TWRVWKVRW SEQ ID NO500 HH501 QRGIVIWRK SEQ ID NO 501 HH502 GKWWKWGIW SEQ ID NO 502 HH503RVRRWWFVR SEQ ID NO 503 HH504 FWRRRVKWR SEQ ID NO 504 HH505 FRRYQNIVRSEQ ID NO 505 HH506 RFWRWIFKW SEQ ID NO 506 HH507 KRNVKRNWK SEQ ID NO507 HH508 WYSLIIFKR SEQ ID NO 508 HH509 RKNRRIRVV SEQ ID NO 509 HH510FFRKRRWRI SEQ ID NO 510 HH511 WKIRKVIKW SEQ ID NO 511 HH512 IKWYWRKKKSEQ ID NO 512 HH513 KRGWRKRWW SEQ ID NO 513 HH514 RKWMGRRIR SEQ ID NO514 HH515 WKGKKRRVI SEQ ID NO 515 HH516 KVIRYKVYI SEQ ID NO 516 HH517RRTRKWILR SEQ ID NO 517 HH518 YNWNWLRRW SEQ ID NO 518 HH519 KWKHWRWQWSEQ ID NO 519 HH520 RKIVVKVRV SEQ ID NO 520 HH521 QYLGWRFKW SEQ ID NO521 HH522 KIKTRKVKY SEQ ID NO 522 HH523 VWIRWRRRW SEQ ID NO 523 HH524WGVRVRRLI SEQ ID NO 524 HH525 WWKRVWKFI SEQ ID NO 525 HH526 YWIYSRLRRSEQ ID NO 526 HH527 RRYWKFKRR SEQ ID NO 527 HH528 IVRRVIIRV SEQ ID NO528 HH529 ARRRGLKVW SEQ ID NO 529 HH530 RRWVRRWWR SEQ ID NO 530 HH531WKWKWKWQS SEQ ID NO 531 HH532 RWKVKQRRR SEQ ID NO 532 HH533 YWTKFRLRISEQ ID NO 533 HH534 WVIKVRIRW SEQ ID NO 534 HH535 ARVQVYKYR SEQ ID NO535 HH536 KWRWHWVYV SEQ ID NO 536 HH537 KVKYKFRRW SEQ ID NO 537 HH538RFRKRKNRI SEQ ID NO 538 HH539 MFRRRFIWK SEQ ID NO 539 HH540 WRLRRFRLWSEQ ID NO 540 HH541 WIQRIRIWV SEQ ID NO 541 HH542 RRYHWRIYI SEQ ID NO542 HH543 SRFWRRWRK SEQ ID NO 543 HH544 YRVWIIRRK SEQ ID NO 544 HH545WRVSWLIWR SEQ ID NO 545 HH546 RFVKRKIVW SEQ ID NO 546 HH547 RIYKIRWIISEQ ID NO 547 HH548 RKFWHRGTI SEQ ID NO 548 HH549 AWVVWRKRW SEQ ID NO549 HH550 WVWGKVRWG SEQ ID NO 550 HH551 FGIRFRRMV SEQ ID NO 551 HH552FWIRKVFRI SEQ ID NO 552 HH553 KRWKVRVVW SEQ ID NO 553 HH554 KIRIWRIWVSEQ ID NO 554 HH555 RGRWKRIKK SEQ ID NO 555 HH556 RLWFLVLRR SEQ ID NO556 HH557 IIRVTRWTK SEQ ID NO 557 HH558 AMWRWKWRK SEQ ID NO 558 HH559TRKYFGRFV SEQ ID NO 559 HH560 ARRVKKKRR SEQ ID NO 560 HH561 RWWKIWKRRSEQ ID NO 561 HH562 RWRYKIQKW SEQ ID NO 562 HH563 RVGIKIKMK SEQ ID NO563 HH564 WVLKLRYKW SEQ ID NO 564 HH565 FRRKWIFKK SEQ ID NO 565 HH566WIQKLWRQR SEQ ID NO 566 HH567 RIVRLHVRK SEQ ID NO 567 HH568 VRIGWRRVKSEQ ID NO 568 HH569 RRRIGIKRF SEQ ID NO 569 HH570 RRRRKKVRI SEQ ID NO570 HH571 KLWRYKRWR SEQ ID NO 571 HH572 RIRRFIKKW SEQ ID NO 572 HH573LWHKKKKIW SEQ ID NO 573 HH574 LTRRFWLRR SEQ ID NO 574 HH575 RRRYVIRRRSEQ ID NO 575 HH576 WGWRWIWIK SEQ ID NO 576 HH577 RWRWQRGRF SEQ ID NO577 HH578 RRKKWKVRI SEQ ID NO 578 HH579 KMKLYKGSM SEQ ID NO 579 HH580GTIRWWRRR SEQ ID NO 580 HH581 SLRRYIWRF SEQ ID NO 581 HH582 GRYWKKWRRSEQ ID NO 582 HH583 WIRQFRWKK SEQ ID NO 583 HH584 AKVRRIKHW SEQ ID NO584 HH585 YSRRKTWWI SEQ ID NO 585 HH586 RGRWWIRRQ SEQ ID NO 586 HH587WVFRWVWWR SEQ ID NO 587 HH588 VYRVWWLKW SEQ ID NO 588 HH589 WWVRRRVGWSEQ ID NO 589 HH590 WFKIKRLYL SEQ ID NO 590 HH591 WKMWKRGWT SEQ ID NO591 HH592 RWWRKSRRL SEQ ID NO 592 HH593 FWRIRWWRW SEQ ID NO 593 HH594VWWFGKRTT SEQ ID NO 594 HH595 VRIIWWIWR SEQ ID NO 595 HH596 WWVRIWRWMSEQ ID NO 596 HH597 RKWKKWFHR SEQ ID NO 597 HH598 RKWKFWGYK SEQ ID NO598 HH599 FWYIWSKRV SEQ ID NO 599 HH600 YWRQFRRKQ SEQ ID NO 600 HH601WWWKVKSRR SEQ ID NO 601 HH602 WRLWIWWIR SEQ ID NO 602 HH603 QFRVNRRKYSEQ ID NO 603 HH604 RYRFWWVRR SEQ ID NO 604 HH605 THIWLRRRR SEQ ID NO605 HH606 RRRFRKRRM SEQ ID NO 606 HH607 LYTRVRRYS SEQ ID NO 607 HH608WSIRRLWWL SEQ ID NO 608 HH609 YKIKRRRYG SEQ ID NO 609 HH610 WKRIQFRRKSEQ ID NO 610 HH611 HKKRRIWRK SEQ ID NO 611 HH612 WRLIRWWIR SEQ ID NO612 HH613 LRKNWWWRR SEQ ID NO 613 HH614 VKRIRIWML SEQ ID NO 614 HH615IRYRNWKWL SEQ ID NO 615 HH616 GRILSRRWK SEQ ID NO 616 HH617 KHWKIHVRWSEQ ID NO 617 HH618 WIYWKVWRR SEQ ID NO 618 HH619 KLWKVRNRR SEQ ID NO619 HH620 RRVYYYKWV SEQ ID NO 620 HH621 WRWGVFRLR SEQ ID NO 621 HH622IWRVLKKRV SEQ ID NO 622 HH623 AKKFWRNWI SEQ ID NO 623 HH624 RQWRKVVKKSEQ ID NO 624 HH625 GWKRWWVML SEQ ID NO 625 HH626 KWRRTRRRK SEQ ID NO626 HH627 FRRMKRFLR SEQ ID NO 627 HH628 RSWNWWWIR SEQ ID NO 628 HH629WRRRIWINR SEQ ID NO 629 HH630 RWKWFYLKR SEQ ID NO 630 HH631 RKRTIWRIISEQ ID NO 631 HH632 RRRVWWRRR SEQ ID NO 632 HH633 KWRFKWWKR SEQ ID NO633 HH634 KWIWGWRRW SEQ ID NO 634 HH635 WIKRKWKMR SEQ ID NO 635 HH636MWKKVLRRV SEQ ID NO 636 HH637 WRWRIFHWL SEQ ID NO 637 HH638 KIQRWKGKRSEQ ID NO 638 HH639 LWYKYWRWR SEQ ID NO 639 HH640 YVRRIWKIT SEQ ID NO640 HH641 RWRQYRSRW SEQ ID NO 641 HH642 VGRWKRRRW SEQ ID NO 642 HH643KSSRIYILF SEQ ID NO 643 HH644 AKWWWYRKI SEQ ID NO 644 HH645 FYWWRWFRVSEQ ID NO 645 HH646 RTRWLRYRR SEQ ID NO 646 HH647 WNIIWWIRR SEQ ID NO647 HH648 KRGFWWWRI SEQ ID NO 648 HH649 RRRKKYIIR SEQ ID NO 649 HH650VWKVGWYYR SEQ ID NO 650 HH651 LKFSTGRVR SEQ ID NO 651 HH652 RRVWVRRKRSEQ ID NO 652 HH653 RFWYMWKYV SEQ ID NO 653 HH654 WYVRWMGRR SEQ ID NO654 HH655 WKRRMRRRK SEQ ID NO 655 HH656 RVLRRVSWV SEQ ID NO 656 HH657RRLRKKWGW SEQ ID NO 657 HH658 WYKKIRLII SEQ ID NO 658 HH659 IYIIIWRTKSEQ ID NO 659 HH660 TWRMRVKVS SEQ ID NO 660 HH661 AWWKIRWRI SEQ ID NO661 HH662 RVRRYRWSW SEQ ID NO 662 HH663 IWRIRRFRI SEQ ID NO 663 HH664KIRRKWWWF SEQ ID NO 664 HH665 RRFWWIKIR SEQ ID NO 665 HH666 WYWWRVRRVSEQ ID NO 666 HH667 WYKLWRRKV SEQ ID NO 667 HH668 WWFSWRWRV SEQ ID NO668 HH669 RFKTRRGWR SEQ ID NO 669 HH670 WIWIVRRRV SEQ ID NO 670 HH671RRFKKWMYW SEQ ID NO 671 HH672 RWYRVIRWK SEQ ID NO 672 HH673 YRWMVRWVRSEQ ID NO 673 HH674 KVRRYNRRR SEQ ID NO 674 HH675 WFVWNRRVV SEQ ID NO675 HH676 RWKWRWRWY SEQ ID NO 676 HH677 ARWRVRKWW SEQ ID NO 677 HH678KIKFWIIRR SEQ ID NO 678 HH679 WYWRVRLQW SEQ ID NO 679 HH680 YWWWKRRRRSEQ ID NO 680 HH681 FIKRVRRRW SEQ ID NO 681 HH682 VSVVFRRRY SEQ ID NO682 HH683 KFRVMVRVL SEQ ID NO 683 HH684 WMYYKRRRR SEQ ID NO 684 HH685IWIWWRWRW SEQ ID NO 685 HH686 WKKKKIIRV SEQ ID NO 686 HH687 RRGWRRRRRSEQ ID NO 687 HH688 WRWRKIWKW SEQ ID NO 688 HH689 WWRWKRRII SEQ ID NO689 HH690 WKVRWKIRR SEQ ID NO 690 HH691 RFWVRGRRS SEQ ID NO 691 HH692RRWVLWRRR SEQ ID NO 692 HH693 KYIWKKRRY SEQ ID NO 693 HH694 KWQWIRKIRSEQ ID NO 694 HH695 YWIRRRWRL SEQ ID NO 695 HH696 RVKWIKWLH SEQ ID NO696 HH697 YVRQWKKRR SEQ ID NO 697 HH698 WKIVGVFRV SEQ ID NO 698 HH699VIKYVRMWW SEQ ID NO 699 HH700 RRRRVWRVR SEQ ID NO 700 HH701 RRRKIRVYRSEQ ID NO 701 HH702 RRNRWRRIR SEQ ID NO 702 HH703 IRKWIWRRV SEQ ID NO703 HH704 QRWRVRRRY SEQ ID NO 704 HH705 WWMIIKIRN SEQ ID NO 705 HH706ARRRGRRVM SEQ ID NO 706 HH707 RRWHWRKRK SEQ ID NO 707 HH708 KRFLRKRRFSEQ ID NO 708 HH709 RWKGWYLRT SEQ ID NO 709 HH710 WSWRGRRKF SEQ ID NO710 HH711 KIIMKRRRW SEQ ID NO 711 HH712 VWKRFLHWR SEQ ID NO 712 HH713RLKRRKKWR SEQ ID NO 713 HH714 AVRKFRRVT SEQ ID NO 714 HH715 IKQRFWWRTSEQ ID NO 715 HH716 WKIVVWIIK SEQ ID NO 716 HH717 LYRWIVWKR SEQ ID NO717 HH718 WWWRWRIRK SEQ ID NO 718 HH719 RLWRKWQWN SEQ ID NO 719 HH720RVKLRWGWR SEQ ID NO 720 HH721 AWRYKRRIF SEQ ID NO 721 HH722 KRWQIRGITSEQ ID NO 722 HH723 KRWRWRWRW SEQ ID NO 723 HH724 KRWVYKYRV SEQ ID NO724 HH725 VHWRWRFWK SEQ ID NO 725 HH726 FVGKTKRKR SEQ ID NO 726 HH727RLRFGWFLF SEQ ID NO 727 HH728 AKRWIWIQV SEQ ID NO 728 HH729 RKYVRRWVYSEQ ID NO 729 HH730 YRVYWWWWR SEQ ID NO 730 HH731 KRRKKRRVR SEQ ID NO731 HH732 KKVRFTITW SEQ ID NO 732 HH733 KLWYWKKVV SEQ ID NO 733 HH734WRWGLRWWQ SEQ ID NO 734 HH735 AFFYRWWIR SEQ ID NO 735 HH736 WYWRRRRLKSEQ ID NO 736 HH737 YKFRWRIYI SEQ ID NO 737 HH738 WLRKVWNWR SEQ ID NO738 HH739 RVRFKVYRV SEQ ID NO 739 HH740 RWLSKIWKV SEQ ID NO 740 HH741RRRLGWRRG SEQ ID NO 741 HH742 KKWGGGLVK SEQ ID NO 742 HH743 YWWLWRKKRSEQ ID NO 743 HH744 WIRLWVKWR SEQ ID NO 744 HH745 GRRSTHWRI SEQ ID NO745 HH746 KKKLFINTW SEQ ID NO 746 HH747 VYRRRRVKG SEQ ID NO 747 HH748KGWIIWKIV SEQ ID NO 748 HH749 VFHRIRRIK SEQ ID NO 749 HH750 RLRLWKSKRSEQ ID NO 750 HH751 RRKVFKLRR SEQ ID NO 751 HH752 VWLKVYWFK SEQ ID NO752 HH753 VRWGRRRWV SEQ ID NO 753 HH754 RYNWVRRKK SEQ ID NO 754 HH755KIRWRKYHL SEQ ID NO 755 HH756 VIWRWRKFY SEQ ID NO 756 HH757 RRWWKWWWRSEQ ID NO 757 HH758 WRVKGKRSK SEQ ID NO 758 HH759 RWRTRRNIV SEQ ID NO759 HH760 WWFSIRLWR SEQ ID NO 760 HH761 YTWYIKKKR SEQ ID NO 761 HH762VWRRKKYWR SEQ ID NO 762 HH763 YLTRFVKYF SEQ ID NO 763 HH764 KRWKHIRRISEQ ID NO 764 HH765 WIVWIRKRI SEQ ID NO 765 HH766 RRWVIRIYK SEQ ID NO766 HH767 WFWRRKMIR SEQ ID NO 767 HH768 RYRRWVRKR SEQ ID NO 768 HH769RKWWWKWRR SEQ ID NO 769 HH770 RIWMFKIFR SEQ ID NO 770 HH771 IVRVGIFRLSEQ ID NO 771 HH772 IIRLIKWWR SEQ ID NO 772 HH773 WVRRYQMRR SEQ ID NO773 HH774 WQVVMRYRR SEQ ID NO 774 HH775 KKWKVWRFG SEQ ID NO 775 HH776WRYWWTRRI SEQ ID NO 776 HH777 RIRKGWKWG SEQ ID NO 777 HH778 KKRRGNRVRSEQ ID NO 778 HH779 VMRKLRRRW SEQ ID NO 779 HH780 RNRTHWWRK SEQ ID NO780 HH781 RFTWWWRKF SEQ ID NO 781 HH782 KRIRYKRWH SEQ ID NO 782 HH783RWRRYGRVY SEQ ID NO 783 HH784 TVVKKRVKK SEQ ID NO 784 HH785 RKYRRRYRRSEQ ID NO 785 HH786 YFRWWKRWI SEQ ID NO 786 HH787 WWQWIVWRK SEQ ID NO787 HH788 RKRLYRWIK SEQ ID NO 788 HH789 GWWKNWRWW SEQ ID NO 789 HH790KWWWYWYRR SEQ ID NO 790 HH791 RFKWFIRRF SEQ ID NO 791 HH792 RIRRLWNIVSEQ ID NO 792 HH793 ARWMWRRWR SEQ ID NO 793 HH794 LVRWVWGKR SEQ ID NO794 HH795 KRWLKWWRV SEQ ID NO 795 HH796 FVYRGWRRK SEQ ID NO 796 HH797RRRWKIYKW SEQ ID NO 797 HH798 KRWWQWRWF SEQ ID NO 798 HH799 KRVKVRWVTSEQ ID NO 799 HH800 RFKYWRWWQ SEQ ID NO 800 HH801 KRQWWRVFK SEQ ID NO801 HH802 FKIVWWRRR SEQ ID NO 802 HH803 QWWWKYRWK SEQ ID NO 803 HH804RWLRIRKVY SEQ ID NO 804 HH805 RYKRVVYRH SEQ ID NO 805 HH806 KVRWKWWGWSEQ ID NO 806 HH807 IWKVRIFKR SEQ ID NO 807 HH808 AIWHKTRRL SEQ ID NO808 HH809 IRQRVRWRW SEQ ID NO 809 HH810 MKVWIRWRI SEQ ID NO 810 HH811QRRWWGRFK SEQ ID NO 811 HH812 NKRVWFIYR SEQ ID NO 812 HH813 RVVNWKGGLSEQ ID NO 813 HH814 RYRRFRVRW SEQ ID NO 814 HH815 KKVRRVIWW SEQ ID NO815 HH816 WFTRWKWRW SEQ ID NO 816 HH817 KWVWFRWRK SEQ ID NO 817 HH818KYLRSVIFY SEQ ID NO 818 HH819 FKRSWVQIV SEQ ID NO 819 HH820 RWWFIRKWWSEQ ID NO 820 HH821 IRRWKRVWW SEQ ID NO 821 HH822 QKWYRQRRN SEQ ID NO822 HH823 VWRKWYRVK SEQ ID NO 823 HH824 KKKLWRKFR SEQ ID NO 824 HH825RRWWWWRFN SEQ ID NO 825 HH826 WFFKSKVYW SEQ ID NO 826 HH827 RVVNLNWRWSEQ ID NO 827 HH828 RWRRNWMTK SEQ ID NO 828 HH829 WKIWKIRWF SEQ ID NO829 HH830 WWFWVIRKY SEQ ID NO 830 HH831 RYVKIRWVR SEQ ID NO 831 HH832RIWILSWRW SEQ ID NO 832 HH833 KSWRKLFIW SEQ ID NO 833 HH834 VWVRWKIWYSEQ ID NO 834 HH835 KKRRFKRRY SEQ ID NO 835 HH836 RFWKKIRRH SEQ ID NO836 HH837 RKVWWRVFY SEQ ID NO 837 HH838 YWRRKWRRK SEQ ID NO 838 HH839KRIRRWKWW SEQ ID NO 839 HH840 YWRYLWIRF SEQ ID NO 840 HH841 IIYKWRWYWSEQ ID NO 841 HH842 QTVYLIFRR SEQ ID NO 842 HH843 AKKIKWLVW SEQ ID NO843 HH844 YRFVRRWIV SEQ ID NO 844 HH845 VWRRYWWYR SEQ ID NO 845 HH846ARKWKYWRF SEQ ID NO 846 HH847 RKRVIKRWR SEQ ID NO 847 HH848 RSFWWMWFKSEQ ID NO 848 HH849 WRINIFKRI SEQ ID NO 849 HH850 RWRVLKRRK SEQ ID NO850 HH851 RWWVIWWWK SEQ ID NO 851 HH852 KLIRIWWWW SEQ ID NO 852 HH853FKRKRWWGI SEQ ID NO 853 HH854 VWHWWRWRW SEQ ID NO 854 HH855 WKRWLIIGRSEQ ID NO 855 HH856 AYRWWTRFK SEQ ID NO 856 HH857 SWWWIWLKK SEQ ID NO857 HH858 FVIWKYIRV SEQ ID NO 858 HH859 RWVRTRRRR SEQ ID NO 859 HH860RRSWWYKRR SEQ ID NO 860 HH861 RKYVWWKSI SEQ ID NO 861 HH862 WWKRYIVKKSEQ ID NO 862 HH863 WFIRVWRYR SEQ ID NO 863 HH864 WKMWLRKHW SEQ ID NO864 HH865 RRFFWKKGI SEQ ID NO 865 HH866 KRWTFWSRR SEQ ID NO 866 HH867AVQRWRWVV SEQ ID NO 867 HH868 IWKYGWRYK SEQ ID NO 868 HH869 IIKWWRRWRSEQ ID NO 869 HH870 AFRKVKRWG SEQ ID NO 870 HH871 MGFTRKWQF SEQ ID NO871 HH872 NWIRWRKWR SEQ ID NO 872 HH873 RIGRKLRIR SEQ ID NO 873 HH874RWWRWRHVI SEQ ID NO 874 HH875 RLVSKRRRK SEQ ID NO 875 HH876 RRKYWKKYRSEQ ID NO 876 HH877 IILWWYRRK SEQ ID NO 877 HH878 IYFWWWRIR SEQ ID NO878 HH879 HKRKWWRFR SEQ ID NO 879 HH880 IGRFWRRWL SEQ ID NO 880 HH881RIRRVLVYV SEQ ID NO 881 HH882 WWLRGRRWL SEQ ID NO 882 HH883 VRIRKRRWRSEQ ID NO 883 HH884 WWRRKWWRR SEQ ID NO 884 HH885 WWWRSFRKR SEQ ID NO885 HH886 VGQKWRKRT SEQ ID NO 886 HH887 FRRRYRVYR SEQ ID NO 887 HH888RIRRKRKGR SEQ ID NO 888 HH889 WKWVTRMYI SEQ ID NO 889 HH890 KVVRKKRLRSEQ ID NO 890 HH891 RKRRKHWRY SEQ ID NO 891 HH892 RVTRTWQRW SEQ ID NO892 HH893 RRRITRKRI SEQ ID NO 893 HH894 RLILIKKKW SEQ ID NO 894 HH895WKRRWSRSR SEQ ID NO 895 HH896 MWWWFLWRR SEQ ID NO 896 HH897 RWVRIWKKKSEQ ID NO 897 HH898 KRRVWRMWR SEQ ID NO 898 HH899 WHWWIRWWR SEQ ID NO899 HH900 WWRRLRWLV SEQ ID NO 900 HH901 KWWIWKRRR SEQ ID NO 901 HH902RYGRKWMIW SEQ ID NO 902 HH903 RVKKIKLFI SEQ ID NO 903 HH904 RIRYIQRVWSEQ ID NO 904 HH905 RLIRWWRKR SEQ ID NO 905 HH906 QRGRWLRRG SEQ ID NO906 HH907 RRRRWIRKK SEQ ID NO 907 HH908 LGRRWRYRR SEQ ID NO 908 HH909FKIVHVKVR SEQ ID NO 909 HH910 FRKKYRVRR SEQ ID NO 910 HH911 WKYKYRIRLSEQ ID NO 911 HH912 HVRRWWRII SEQ ID NO 912 HH913 RFKWWRRYW SEQ ID NO913 HH914 RRRRMRKKI SEQ ID NO 914 HH915 RRIRGRVGR SEQ ID NO 915 HH916AFWRWIRFK SEQ ID NO 916 HH917 VKKRKIVIY SEQ ID NO 917 HH918 KRVKWTWRKSEQ ID NO 918 HH919 TGVGRGYRI SEQ ID NO 919 HH920 LSWKWWRRV SEQ ID NO920 HH921 IKTFIKRWR SEQ ID NO 921 HH922 KMRLKWKRR SEQ ID NO 922 HH923WRWYVTRRK SEQ ID NO 923 HH924 IYRRRRKLR SEQ ID NO 924 HH925 VWWKWWRWWSEQ ID NO 925 HH926 KYKKGWRVV SEQ ID NO 926 HH927 KWRRWYYWR SEQ ID NO927 HH928 RRWVFGRRY SEQ ID NO 928 HH929 GFTWKKKRR SEQ ID NO 929 HH930YKKIRIKRR SEQ ID NO 930 HH931 VWIRRIKRR SEQ ID NO 931 HH932 WWKWIRKIVSEQ ID NO 932 HH933 WRRKWWSRW SEQ ID NO 933 HH934 VTRRRTRIK SEQ ID NO934 HH935 RKRWFVYIW SEQ ID NO 935 HH936 IIKWKRIMI SEQ ID NO 936 HH937FNRWWWKKI SEQ ID NO 937 HH938 RYKSRRVRR SEQ ID NO 938 HH939 VKVIKKFVRSEQ ID NO 939 HH940 KWKWLQGRR SEQ ID NO 940 HH941 KVRWWYNIK SEQ ID NO941 HH942 FWFRIRKLK SEQ ID NO 942 HH943 KRRKQRKYR SEQ ID NO 943 HH944AKNSKRRLW SEQ ID NO 944 HH945 RNRRIFRYS SEQ ID NO 945 HH946 RWTKWFLVRSEQ ID NO 946 HH947 RIRRTRRTR SEQ ID NO 947 HH948 KIRWWRISI SEQ ID NO948 HH949 YKGRWGRRW SEQ ID NO 949 HH950 MYYRIKQKW SEQ ID NO 950 HH951WRIQRWRWQ SEQ ID NO 951 HH952 IRRWSYRRW SEQ ID NO 952 HH953 VRIWKIIWWSEQ ID NO 953 HH954 RWRWWWLWK SEQ ID NO 954 HH955 TKRRWIWIT SEQ ID NO955 HH956 RRWHYWKGW SEQ ID NO 956 HH957 WRIRKWWMR SEQ ID NO 957 HH958KRRTRWWVR SEQ ID NO 958 HH959 RKWRVWKRR SEQ ID NO 959 HH960 WRVWKIRVRSEQ ID NO 960 HH961 KYWGIGGWR SEQ ID NO 961 HH962 RLISRRRKK SEQ ID NO962 HH963 VSRRIVRRM SEQ ID NO 963 HH964 ITKWWRKRR SEQ ID NO 964 HH965KWKIQLWKI SEQ ID NO 965 HH966 KKWTWWYVI SEQ ID NO 966 HH967 SWKKNRKIWSEQ ID NO 967 HH968 HKRQYRKWF SEQ ID NO 968 HH969 IFKWFYRRK SEQ ID NO969 Bac2A RLARIVVIRVAR SEQ ID NO 970 Indolicidin ILPWKWPWWPWRR SEQ ID NO971 Scrambled VRLRIRVAVIRA SEQ ID NO 972 HH970 ILKWKWPWWKWRR SEQ ID NO973 HH971 ILPWKWRWWKWRR SEQ ID NO 974 HH972 FLPKKFRWWKYRK SEQ ID NO 975HH973 FIKWKFRWWKWRK SEQ ID NO 976 HH974 KWPWWPWRR SEQ ID NO 977 HH975KWPWWPWRK SEQ ID NO 978 HH976 KFPWWPWRR SEQ ID NO 979 HH977 KKPWWPWRRSEQ ID NO 980 HH978 KWRWWPWRR SEQ ID NO 981 HH979 KWPKWPWRR SEQ ID NO982 HH980 KWPWKPWRR SEQ ID NO 983 HH981 KWPWWKWRR SEQ ID NO 984 HH982KWPWWPKRR SEQ ID NO 985 HH983 KWPWWPWRR SEQ ID NO 986 HH984 KFRWWPWRRSEQ ID NO 987 HH985 KFRWWKWRR SEQ ID NO 988 HH986 KWRWWKKRR SEQ ID NO989 HH987 KKKWWKWRR SEQ ID NO 990 HH988 KFHWWIWRK SEQ ID NO 991 HH989KFHWWKWRK SEQ ID NO 992 HH990 KFKWWKYRK SEQ ID NO 993 HH991 KFKFFKYRKSEQ ID NO 994 HH992 KFKFFKFRK SEQ ID NO 995 HH993 PWWPWRR SEQ ID NO 996HH994 KWWPWRR SEQ ID NO 997 HH995 PWWKWRR SEQ ID NO 998 HH996 RWWPWRRSEQ ID NO 999 HH997 PKWPWRR SEQ ID NO 1000 HH998 PWKPWRR SEQ ID NO 1001HH999 PWWKWRR SEQ ID NO 1002 HH1000 PWWPKRR SEQ ID NO 1003 HH1001PWWPWRK SEQ ID NO 1004 HH1002 RWWKWRR SEQ ID NO 1005 HH1003 RWWKWRK SEQID NO 1006 HH1004 RFWKWRR SEQ ID NO 1007 HH1005 RWWIKRR SEQ ID NO 1008HH1006 RWWIYRR SEQ ID NO 1009 HH1007 RFFKFRR SEQ ID NO 1010 HH1008KWWKWKK SEQ ID NO 1011 HH1009 KFFKFKK SEQ ID NO 1012 HHC1 RWRWKRWWW SEQID NO 1013 HHC2 RWRRWKWWW SEQ ID NO 1014 HHC3 RWWRWRKWW SEQ ID NO 1015HHC4 RWRRKWWWW SEQ ID NO 1016 HHC5 RWRWWKRWY SEQ ID NO 1017 HHC6RRKRWWWWW SEQ ID NO 1018 HHC7 RWRIKRWWW SEQ ID NO 1019 HHC8 KIWWWWRKRSEQ ID NO 1020 HHC9 RWRRWKWWL SEQ ID NO 1021 HHC10 KRWWKWIRW SEQ ID NO1022 HHC11 KRWWWWWKR SEQ ID NO 1023 HHC12 IRWWKRWWR SEQ ID NO 1024 HHC13IKRWWRWWR SEQ ID NO 1025 HHC14 RRKWWWRWW SEQ ID NO 1026 HHC15 RKWWRWWRWSEQ ID NO 1027 HHC16 KRWWWWRFR SEQ ID NO 1028 HHC17 IKRWWWRRW SEQ ID NO1029 HHC18 KRWWWVWKR SEQ ID NO 1030 HHC19 KWRRWKRWW SEQ ID NO 1031 HHC20WRWWKIWKR SEQ ID NO 1032 HHC21 WRWRWWKRW SEQ ID NO 1033 HHC22 WKRWKWWKRSEQ ID NO 1034 HHC23 RIKRWWWWR SEQ ID NO 1035 HHC24 IWKRWWRRW SEQ ID NO1036 HHC25 KWWKIWWKR SEQ ID NO 1037 HHC26 RKRWLWRWW SEQ ID NO 1038 HHC27KRWRWWRWW SEQ ID NO 1039 HHC28 KKRWLWWWR SEQ ID NO 1040 HHC29 RWWRKWWIRSEQ ID NO 1041 HHC30 KWWRWWRKW SEQ ID NO 1042 HHC31 KRWWIRWWR SEQ ID NO1043 HHC32 KIWWWWRRR SEQ ID NO 1044 HHC33 RRRKWWIWW SEQ ID NO 1045 HHC34RRRWWWWWW SEQ ID NO 1046 HHC35 RWWIRKWWR SEQ ID NO 1047 HHC36 KRWWKWWRRSEQ ID NO 1048 HHC37 KRWWRKWWR SEQ ID NO 1049 HHC38 RRIWRWWWW SEQ ID NO1050 HHC39 IRRRKWWWW SEQ ID NO 1051 HHC40 KRKIWWWIR SEQ ID NO 1052 HHC41RKIWWWRIR SEQ ID NO 1053 HHC42 KRWWIWRIR SEQ ID NO 1054 HHC43 RWFRWWKRWSEQ ID NO 1055 HHC44 WRWWWKKWR SEQ ID NO 1056 HHC45 WKRWWKKWR SEQ ID NO1057 HHC46 WKRWRWIRW SEQ ID NO 1058 HHC47 WRWWKWWRR SEQ ID NO 1059 HHC48WKKWWKRRW SEQ ID NO 1060 HHC49 WRWYWWKKR SEQ ID NO 1061 HHC50 WRRWWKWWRSEQ ID NO 1062 HHC51 IRMWVKRWR SEQ ID NO 1063 HHC52 RIWYWYKRW SEQ ID NO1064 HHC53 FRRWWKWFK SEQ ID NO 1065 HHC54 RVRWWKKRW SEQ ID NO 1066 HHC55RLKKVRWWW SEQ ID NO 1067 HHC56 RWWLKIRKW SEQ ID NO 1068 HHC57 LRWWWIKRISEQ ID NO 1069 HHC58 TRKVWWWRW SEQ ID NO 1070 HHC59 KRFWIWFWR SEQ ID NO1071 HHC60 KKRWVWVIR SEQ ID NO 1072 HHC61 KRWVWYRYW SEQ ID NO 1073 HHC62IRKWRRWWK SEQ ID NO 1074 HHC63 RHWKTWWKR SEQ ID NO 1075 HHC64 RRFKKWYWYSEQ ID NO 1076 HHC65 RIKVIWWWR SEQ ID NO 1077 HHC66 RKRLKWWIY SEQ ID NO1078 HHC67 LVFRKYWKR SEQ ID NO 1079 HHC68 RRRWWWIIV SEQ ID NO 1080 HHC69KKRWVWIRY SEQ ID NO 1081 HHC70 RWRIKFKRW SEQ ID NO 1082 HHC71 KWKIFRRWWSEQ ID NO 1083 HHC72 IWKRWRKRL SEQ ID NO 1084 HHC73 RRRKWWIWG SEQ ID NO1085 HHC74 RWLVLRKRW SEQ ID NO 1086 HHC75 RKWIWRWFL SEQ ID NO 1087 HHC76KRRRIWWWK SEQ ID NO 1088 HHC77 IWWKWRRWV SEQ ID NO 1089 HHC78 LRWRWWKIKSEQ ID NO 1090 HHC79 RWKMWWRWV SEQ ID NO 1091 HHC80 VKRYYWRWR SEQ ID NO1092 HHC81 RWYRKRWSW SEQ ID NO 1093 HHC82 KRKLIRWWW SEQ ID NO 1094 HHC83RWRWWIKII SEQ ID NO 1095 HHC84 KFRKRVWWW SEQ ID NO 1096 HHC85 IWIWRKLRWSEQ ID NO 1097 HHC86 LRFILWWKR SEQ ID NO 1098 HHC87 RVWFKRRWW SEQ ID NO1099 HHC88 RRWFVKWWY SEQ ID NO 1100 HHC89 KWWLVWKRK SEQ ID NO 1101 HHC90RWILWWWRI SEQ ID NO 1102 HHC91 KRWLTWRFR SEQ ID NO 1103 HHC92 RKWRWRWLKSEQ ID NO 1104 HHC93 IRRRWWWIV SEQ ID NO 1105 HHC94 IKWWWRMRI SEQ ID NO1106 HHC95 RWKIFIRWW SEQ ID NO 1107 HHC96 IRQWWRRWW SEQ ID NO 1108 HHC97RRRKTWYWW SEQ ID NO 1109 HHC98 RRWWHLWRK SEQ ID NO 1110 HHC99 RRWWMRWWVSEQ ID NO 1111 HHC100 RRFKFIRWW SEQ ID NO 1112 HHC101 INRKRRLRW SEQ IDNO 1113 HHC102 RRMKKLRRK SEQ ID NO 1114 HHC103 RKVRWKIRV SEQ ID NO 1115HHC104 VRIVRVRIR SEQ ID NO 1116 HHC105 IKRVKRRKR SEQ ID NO 1117 HHC106RVKTWRVRT SEQ ID NO 1118 HHC107 RVFVKIRMK SEQ ID NO 1119 HHC108IRGRIIFWV SEQ ID NO 1120 HHC109 ATWIWVFRR SEQ ID NO 1121 HHC110KKSKQLWKR SEQ ID NO 1122 HHC111 MINRVRLRW SEQ ID NO 1123 HHC112GGIRRLRWY SEQ ID NO 1124 HHC113 RLVHWIRRV SEQ ID NO 1125 HHC114AWKIKKGRI SEQ ID NO 1126 HHC115 FVVMKRIVW SEQ ID NO 1127 HHC116GIKWRSRRW SEQ ID NO 1128 HHC117 RWMVSKIWY SEQ ID NO 1129 HHC118IVVRVWVVR SEQ ID NO 1130 HHC119 RWIGVIIKY SEQ ID NO 1131 HHC120WIRKRSRIF SEQ ID NO 1132 HHC121 GWKILRKRK SEQ ID NO 1133 HHC122YQRLFVRIR SEQ ID NO 1134 HHC123 AVWKFVKRV SEQ ID NO 1135 HHC124IRKKRRRWT SEQ ID NO 1136 HHC125 ILRVISKRR SEQ ID NO 1137 HHC126AWRFKNIRK SEQ ID NO 1138 HHC127 HYKFQRWIK SEQ ID NO 1139 HHC128RRIRRVRWG SEQ ID NO 1140 HHC129 VLVKKRRRR SEQ ID NO 1141 HHC130RWRGIVHIR SEQ ID NO 1142 HHC131 WRNRKVVWR SEQ ID NO 1143 HHC132KFWWWNYLK SEQ ID NO 1144 HHC133 KRIMKLKMR SEQ ID NO 1145 HHC134IRRRKKRIK SEQ ID NO 1146 HHC135 RKWMGRFLM SEQ ID NO 1147 HHC136RRVQRGKWW SEQ ID NO 1148 HHC137 WHGVRWWKW SEQ ID NO 1149 HHC138WVRFVYRYW SEQ ID NO 1150 HHC139 RKRTKVTWI SEQ ID NO 1151 HHC140IRRIVRRKI SEQ ID NO 1152 HHC141 KIRRKVRWG SEQ ID NO 1153 HHC142AIRRWRIRK SEQ ID NO 1154 HHC143 WRFKVLRQR SEQ ID NO 1155 HHC144RSGKKRWRR SEQ ID NO 1156 HHC145 FMWVYRYKK SEQ ID NO 1157 HHC146RGKYIRWRK SEQ ID NO 1158 HHC147 WVKVWKYTW SEQ ID NO 1159 HHC148VVLKIVRRF SEQ ID NO 1160 HHC149 GKFYKVWVR SEQ ID NO 1161 HHC150SWYRTRKRV SEQ ID NO 1162 HHC151 KNRGRWFSH SEQ ID NO 1163 HHC152AFRGSRHRM SEQ ID NO 1164 HHC153 GRNGWYRIN SEQ ID NO 1165 HHC154AGGMRKRTR SEQ ID NO 1166 HHC155 ATRKGYSKF SEQ ID NO 1167 HHC156SSGVRWSWR SEQ ID NO 1168 HHC157 RVWRNGYSR SEQ ID NO 1169 HHC158WGRTRWSSR SEQ ID NO 1170 HHC159 GKRVWGRGR SEQ ID NO 1171 HHC160SFNWKRSGK SEQ ID NO 1172 HHC161 WGRGGWTNR SEQ ID NO 1173 HHC162ANRWGRGIR SEQ ID NO 1174 HHC163 WGGHKRRGW SEQ ID NO 1175 HHC164WMGGQKWRK SEQ ID NO 1176 HHC165 FVWQKGTNR SEQ ID NO 1177 HHC166HGVWGNRKR SEQ ID NO 1178 HHC167 TRGWSLGTR SEQ ID NO 1179 HHC168GRRVMNQKR SEQ ID NO 1180 HHC169 RNKFGGNWR SEQ ID NO 1181 HHC170GVRVQRNSK SEQ ID NO 1182 HHC171 NQKWSGRRR SEQ ID NO 1183 HHC172RQNGVWRVF SEQ ID NO 1184 HHC173 GRMRLWNGR SEQ ID NO 1185 HHC174WHYRSQVGR SEQ ID NO 1186 HHC175 GWNTMGRRW SEQ ID NO 1187 HHC176RRMGNGGFR SEQ ID NO 1188 HHC177 SKNVRTWRQ SEQ ID NO 1189 HHC178ARGRWINGR SEQ ID NO 1190 HHC179 GSRRSVWVF SEQ ID NO 1191 HHC180WSQNVRTRI SEQ ID NO 1192 HHC181 GMRRWRGKN SEQ ID NO 1193 HHC182RGRTSNWKM SEQ ID NO 1194 HHC183 GRRWGMGVR SEQ ID NO 1195 HHC184WGKRRGWNT SEQ ID NO 1196 HHC185 AMLGGRQWR SEQ ID NO 1197 HHC186QRNKGLRHH SEQ ID NO 1198 HHC187 ARGKSIKNR SEQ ID NO 1199 HHC188NRRNGQMRR SEQ ID NO 1200 HHC189 RGRRQIGKF SEQ ID NO 1201 HHC190ASKRVGVRN SEQ ID NO 1202 HHC191 GRIGGKNVR SEQ ID NO 1203 HHC192NKTGYRWRN SEQ ID NO 1204 HHC193 VSGNWRGSR SEQ ID NO 1205 HHC194GWGGKRRNF SEQ ID NO 1206 HHC195 KNNRRWQGR SEQ ID NO 1207 HHC196GRTMGNGRW SEQ ID NO 1208 HHC197 GRQISWGRT SEQ ID NO 1209 HHC198GGRGTRWHG SEQ ID NO 1210 HHC199 GVRSWSQRT SEQ ID NO 1211 HHC200GSRRFGWNR SEQ ID NO 1212 1001 LVRAIQVRAVIR SEQ ID NO 1213 1002VQRWLIVWRIRK SEQ ID NO 1214 1003 IVWKIKRWWVGR SEQ ID NO 1215 1004RFWKVRVKYIRF SEQ ID NO 1216 1005 VQLRIRVAV SEQ ID NO 1217 1006 VQLRIWVRRSEQ ID NO 1218 1007 WNRVKWIRR SEQ ID NO 1219 1008 RIKWIVRFR SEQ ID NO1220 1009 AIRVVRARLVRR SEQ ID NO 1221 1010 IRWRIRVWVRRI SEQ ID NO 12221011 RRWVVWRIVQRR SEQ ID NO 1223 1012 IFWRRIVIVKKF SEQ ID NO 1224 1013VRLRIRVAV SEQ ID NO 1225 1014 RQVIVRRW SEQ ID NO 1226 1015 VLIRWNGKK SEQID NO 1227 1016 LRIRWIFKR SEQ ID NO 1228 1017 KRIVRRLVARIV SEQ ID NO1229 1018 VRLIVAVRIWRR SEQ ID NO 1230 1019 IVVWRRQLVKNK SEQ ID NO 12311020 VRLRIRWWVLRK SEQ ID NO 1232 1021 VRLRIRVAV SEQ ID NO 1233 1022LRIRVIVWR SEQ ID NO 1234 1023 IRVWVLRQR SEQ ID NO 1235 1024 RIRVIVLKKSEQ ID NO 1236 1025 RRIVKKFQIVRR SEQ ID NO 1237 1026 VQWRIRVRVIKK SEQ IDNO 1238 1027 KKQVSRVKVWRK SEQ ID NO 1239 1028 LIQRIRVRNIVK SEQ ID NO1240 1029 KQFRIRVRV SEQ ID NO 1241 1030 FRIRVRVIR SEQ ID NO 1242 1031WRWRVRVWR SEQ ID NO 1243 1032 IRVRVIWRK SEQ ID NO 1244 1033 RRVIVKKFRIRRSEQ ID NO 1245 1034 KQFRNRLRIVKK SEQ ID NO 1246 1035 KRWRWIVRNIRR SEQ IDNO 1247 1036 VQFRIRVIVIRK SEQ ID NO 1248 1037 KRFRIRVRV SEQ ID NO 12491038 IVVRRVIRK SEQ ID NO 1250 1039 IWVIRRVWR SEQ ID NO 1251 1040FQVVKIKVR SEQ ID NO 1252 1041 VIWIRWR SEQ ID NO 1253 1042 IVWIWRR SEQ IDNO 1254 1043 WIVIWRR SEQ ID NO 1255 1044 RRWIVWI SEQ ID NO 1256 1045RWWRIVI SEQ ID NO 1257 1046 WIRVIRW SEQ ID NO 1258 1047 IIRRWWV SEQ IDNO 1259 1048 IRWVIRW SEQ ID NO 1260 HH1010 ILRWKWRWWRWRR SEQ ID NO 1261HH1011 RWRWWRWRR SEQ ID NO 1262 HH1012 KWKWWKWKK SEQ ID NO 1263 HH1013RWWRWRR SEQ ID NO 1264

Minimal inhibitory concentration (MIC) determination. The MIC of thepeptides were measured using a modified broth microdilution method inMueller Hinton (MH) medium (Wu M, Hancock R E W. 1999. Interaction ofthe cyclic antimicrobial cationic peptide bactenecin with the outer andcytoplasmic membrane. J Biol Chem 274, 29-35). Briefly, the peptideswere dissolved and stored in glass vials. The assay was performed insterile 96-well polypropylene microtitre plates were used. Serialdilutions of the peptides to be assayed were performed in 0.01% aceticacid (Fisher) containing 0.2% bovine serum albumin (Boehringer MannheimGmbH) at 10× the desired final concentration. Ten microlitres of the 10×peptides were added to each well of a 96-well polypropylene platecontaining 90 μl of MH media per well. Bacteria were added to the platefrom an overnight culture at 2-7×10⁵ colony forming units/ml andincubated over night at 37° C. The MIC was taken as the concentration atwhich no growth was observed.

Luminescence-based MIC assay for the non-cleaved peptides on cellulosemembranes. The method followed was as previously described (Hilpert K,Volkmer-Engert R, Walter T, Hancock R E W. High-throughput generation ofsmall antibacterial peptides with improved activity. Nature Biotech23:1008-1012, 2005). Peptides were robotically synthesized on cellulosesheets and then the peptide spots were punched out and transferred to a96 well microtiter plate with a clear polystyrene bottom and opaquewhite polypropylene sides (PerkinElmer, Boston, USA). The spots werewashed two times with 100% ethanol for 5 min and afterwards equilibratedfive times with 100 mM Tris buffer pH 7.3 for 5 min. An overnightculture of Pseudomonas aeruginosa strain H1001 fliC::luxCDABE wasdiluted 1:50 in new MH medium and incubated at 37° C. to an OD600 of0.35. This bacterial culture was diluted 1:25 into 100 mM Tris-HClbuffer, pH 7.3 containing 20 mM glucose. Fifty μl of this culture wasadded to all wells of the microtiter plate and incubated at 37° C. Theluminescence of the strain produced by the FMN-dependent luciferasesystem was detected in a time dependent manner using a Tecan SpectraFluor plus (Tecan, Austria). At the end of the experiment, the membraneswere cleaned by washing the spots two times with 100% ethanol for fiveminutes. After removing the ethanol the membrane was air-dried.

Assay for the cleaved peptides from cellulose support. The peptides werecleaved from the dried membrane in an ammonia atmosphere overnight,resulting in free peptides with an amidated C-terminus. The freepeptides contained two β-alanines at the C-terminus, in addition tobeing amidated due to the linker between the cellulose membrane and thepeptide sequence. The peptide spots were punched out and transferred ina 96-well microtiter plate. Serial dilutions were carried out from themembrane spots. Four rows were filled with four controls including2×Bac2A and 2× an unrelated peptide. The other eight rows were used forserial dilution steps of the peptide variants. An overnight culture ofPseudomonas aeruginosa strain H1001 was diluted 1:500 using either LBmedia or 100 mM Tris buffer pH 7.3, 20 mM glucose and was added to thewells (100 μl/well) containing the peptide spots. In all other wells 50μl were added. The microtiter plate was incubated for 30 min at 37° C.to release the peptides from the membrane. Subsequently, a dilutionseries were performed and the plate was incubated at 37° C. Theluminescence produced by the FMN dependent luciferase system weredetected in a time dependent manner using a Tecan Spectra Fluor plus.

Cytotoxicity and TNFα suppression assay. THP1 cells were cultured inRPMI 1640 medium (supplemented with 10% (v/v) FCS, 1% L-glutamine, and 1nM sodium pyruvate) in E-toxa-clean (Sigma-Aldrich, Oakville, Ontario,Canada)-washed, endotoxin-free bottle. THP1 cells were counted and 250μl per well of 2-4 104 cells/ml were transferred into a 96 well tissuecultured treated polystyrene microtiterplate (Beckton Dickinson,Franklin Lakes, USA). In addition PMA were added (1.3 nM) and the cellswere incubated for three days. After three days the medium wereexchanged and Pseudomonas aeruginosa LPS and the peptides were added.The incubation time was four hours and the supernatant was frozen at−20° C. The cells were stained with Tryphan Blue for 2 minutes andwashed with PBS two times. The viability of the cells was determined bycounting the stained cells over the unstained. The supernatent was usedto measure the TNFα production by an ELISA (eBioscience, San Diego, USA)following the manufactures protocol.

Strains. For the killing assay a mini-Tn5-lux mutant in Pseudomonasaeruginosa H103 was used. The strain is called H1001 and contains afliC::luxCDABE transcriptional fusion resulting in constitutiveexpression of luciferase. The bacterial strains used for theantimicrobial activity assays included Escherichia coli UB1005 (F-,nalA37, metB1), a wild-type Salmonella enterica ssp. typhimurium (S.typhimurium), wild-type Pseudomonas aeruginosa PAO1 strain H103,Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, and aclinical isolate of Staphylococcus epidermidis obtained from Dr. D.Speert (Department of Medicine, University of British Columbia).Antifungal activity was tested using a lab isolate of Candida albicansobtained from Dr. B. Dill (Department of Microbiology and Immunology,University of British Columbia).

Example 2

12-Mer Peptides.

Bactenecin is a short peptide of 12 amino acids with a maximal length ofabout 55 Å. This peptide can kill both Gram positive and Gram negativebacteria. We previously made many scrambled and single amino acidsubstitution mutants of this peptide. A complete substitution analysisof a scrambled variant of Bac2A, SEQ ID NO 972 was synthesized (FIG. 2)identifying peptides that had distinct advantages over their parentpeptide and positionally defining advantageous substitutions. The mostfavoured residues were:

-   -   AA₁=all except D and E    -   AA₂=F,H,K,L,Q,R,S,T,V,Y    -   AA₃=F,W    -   AA₄=K,R    -   AA₅=F,L,M,V,W    -   AA₆=K,R    -   AA₇=V,I    -   AA₈=H,K,N,Q,R,S,Y    -   AA₉=V,M    -   AA₁₀=I,K,R    -   AA₁₁=K,R,H    -   AA₁₂=H,K,N,R,T        It is clear that some amino acids particularly R, K and W were        often preferred to the parent residue. In contrast, some        residues were usually detrimental to activity, namely the acidic        amino acids D and E. Overall substitutions were rarely        conservative and predictable just from the obvious substitution        of e.g. one hydrophobic residue for another. Some positions were        particularly rich candidates for substitution, namely positions        2, 5, 8 and 12 while others were very difficult to improve        especially the charged residues and isoleucin and valin in the        core region. Regarding unfavourable substitutions, the least        favourable substitutions were:    -   AA₁=D,E    -   AA₂=D,E    -   AA₃=D,E    -   AA₄=D,E,F,G,I,Y    -   AA₅=all except F,L,M,V,W,Y    -   AA₆=all except K,R    -   AA₇=all except F,I,L,M,R,V,W,Y    -   AA₈=D,E,P    -   AA₉=D,E,G,P,Q    -   AA₁₀=none    -   AA₁₁=D,E,F,I,L,M,Q,T,V,W    -   AA₁₂=none

A range of novel peptides that are very distinct but possess thematicsimilarities to linear bactenecin (Bac2A) were synthesized and testedfor activity (Table 3).

TABLE 3 Determination of the minimal inhibitory concentrations (MIC)in Mueller-Hinton media for 6 different bacteria and the yeastCandida albicans. The values are averages ofthree independent measurements. Sequence (all C- terminallyMIC (μg/ml)^(a) amidated) Name P. aerug E. coli S. typhi S. aureusS. epi. E. faecalis C. albicans RLARIVVIRVAR Bac2A 50 17 34 17 4 17 9QRLRIRVAVIRA HH1 50 6 25 50 12 50 25 VQLRIRVAVIRA HH2 >50 6 12 50 12 5025 VRFRIRVAVIRA HH3 6 1.6 6 12 1.6 25 6 VRWRIRVAVIRA HH4 6 1.6 6 12 1.612 12 VRLWIRVAVIRA HH5 >50 6 >50 50 3 25 50 VRLRIRVWVIRA HH6 12 3 12 61.6 6 25 VRLRIRVAVRRA HH7 12 6 6 12 1.6 25 6 VRLRIRVAVIRK HH8 6 3 6 61.6 12 3 VQLRIRVRVIRK HH9 6 3 6 12 1.6 12 6 KRFRIRVAVRRA HH10 25 6 >5025 3.1 >50 12 VRLRIRVRVIRK HH11 3 1.6 6 6 0.8 12 3 KQFRIRVRVIRK HH12 6 36 12 1.6 25 6 HQFRFRFRVRRK HH13 50 25 12 12 6 >50 >50 HQWRIRVAVR HH14 506 25 25 12 >50 25 RH KRFRIRVRVIRK HH15 6 1.6 3 6 0.8 25 12 KRWRIRVRVIRKHH16 3 1.6 3 3 0.8 12 6 KIWVRWK-NH2 HH17 >50 50 >50 >50 >50 >50 >50IWVIWRR-NH2 HH18 50 6 12.5 25 6 25 25

As can be seen from Table 3, each of the peptides, except HH17,represents an improvement on the parent peptide. Some, in particularHH8, HH9, HH11, HH12, HH15 and HH16, have excellent broad spectrumantimicrobial activity. For example HH8 and HH11 represent two of thebest anti-Candidal peptides ever identified. HH3, HH4, HH15 and HH16have excellent activity against the major nosocomial Gram negativepathogen E. coli. These results thus show that it is possible to furtheroptimize scrambled peptides and therefore gain peptides with totallydifferent sequences as the original peptide Bac2A.

Example 3

Complete Substitution Analysis of Indolicidin

Peptide synthesis on cellulose is a very effective and inexpensive wayto investigate many different peptide variants for one particularactivity. However, one of the problems of this technique is the lowpeptide amount synthesized on the membrane, about 280 nmol per cm². Anaverage peptide spot used for the experiments presented is about 0.3cm². Therefore, an assay had to be developed that was sensitive enoughto show activity with this amount of peptides. P. aeruginosa H1001 has aluciferase gene cassette incorporated into the bacterial chromosome in agene fliC (involved in flagellar biosynthesis) that is constitutivelyexpressed. It will thus produce light if FMN is present. When thisstrain is killed, e.g. by peptides, the amount of light produced willdecrease due to a decrease in FMN levels in the killed cells. Thisaction can thus be monitored by detecting luminescence in a microtiterplate luminescence reader over time. The volume and amount of cells perwell were optimized for this assay. After screening, we were able tomonitor the killing action with small amounts of the parent peptideBac2A (2 μg/ml of free peptide). Control experiments demonstrated thatthe decrease in luminescence reflected bacterial death as assessed bythe loss of colony forming ability.

To analyze the positional importance of the specific amino acids in thebovine peptide indolicidin, each amino acid was changed to the other 19amino acids one-by-one, creating a total of 247 unique peptides.Activity was assessed in the lux-based assay (FIG. 3). The resultsrevealed definite positional specificity of particular amino acids andmany substitutions that improved the activity of indolicidin.

The most favoured residues were:

-   -   AA₁=F,Y    -   AA₂=F,G,H,I,K,M,P,R    -   AA₃=H,I,K,M,N,Q,R,S    -   AA₄=K,R    -   AA₅=no improvement on K    -   AA₆=F,H,I,K,L,R    -   AA₇=H,K,L,R,S,T    -   AA₈=K,R    -   AA₉=K,R    -   AA₁₀=I,K,R    -   AA₁₁=K,R,Y    -   AA₁₂=K    -   AA₁₃=K

It is clear that some amino acids particularly R and K are oftenpreferred to the parent residue. In contrast, some residues were usuallydetrimental to indolicidin activity, namely the acidic amino acids D andE, while others never led to an improvement in activity, namely A, V andW. Overall substitutions were rarely conservative and predictable justfrom the obvious substitution of e.g. one hydrophobic residue foranother. Some positions were particularly rich candidates forsubstitution, namely positions 2, 3, 6 and 7 while others were verydifficult to improve especially the charged residues.

-   -   Regarding unfavourable substitutions, the least favourable        substitutions were:    -   AA₁=none    -   AA₂=D,E,W    -   AA₃=D,E,F,W,Y    -   AA₄=A,D,E,G,V    -   AA₅=all except K and R    -   AA₆=D,E,Q    -   AA₇=D,E,F    -   AA₈=D,E,L    -   AA₉=D,E    -   AA₁₀=D,E    -   AA₁₁=D,E,I    -   AA₁₂=all except K and R    -   AA₁₃=all except K, R and I

Generally speaking those positions with the most favourablesubstitutions (AA₂, AA₃, AA₆, and AA₇) were the most flexible and hadfew unfavourable substitutions. Three positions with charged residuesdid not readily accept substitutions namely AA₅, AA₁₂, and AA₁₃, and forthese the parent amino acid could only be improved by changing the basicresidue utilized.

The activity of these peptides was confirmed by synthesizing selectedexamples of single and multiple substitutions (Table 3). The majority ofthese had superior activity to the parent peptide indolicidin.

TABLE 4 Antimicrobial activity of single and multiple substitutionvariants of indolicidin (substituted amino acids are indicatedin bold in column 1). MIC (μg/ml)^(a) Sequence Name P. aerug. E. coli.S. typhi S. aureus S. epi. E. faecalis C. albicans ILPWKWPWWPWRRIndolicidin 62 8 31 16 8 31 16 ILPWKFPWWPWRR HH63 62 16 31 8 4 31 16ILKWKWPWWPWRR HH111 16 8 31 8 4 31 8 ILPWKKPWWPWRR HH113 31 31 62 31 8125 62 ILPWKWPWWKWRR HH117 16 8 31 8 2 31 16 ILPWWWPWWPWRRHH235 >84 >84 >84 20 5 84 84 ILKWKWPWWKWRR HH970 16 8 16 8 2 31 16ILPWKWRWWKWRR HH971 16 8 16 8 2 31 8 FLPKKFRWWKYRK HH972 31 16 31 318 >125 31 FIKWKFRWWKWR HH973 8 4 8 4 2 8 8

This was used to synthesize a series of 9 and 7 amino acid peptides andactivity was tested by the luciferase method (Table 5). All synthesized9-mers were active whereas peptides as small as 7 amino acids also hadexcellent antimicrobial activity.

TABLE 5 Antimicrobial activity of selected 9 aminoacid and 7 amino acid peptides. Antimicrobial Name ID Peptide sequenceactivity* HH974 SEQ ID NO 977 KWPWWPWRR +/++ HH975 SEQ ID NO 978KWPWWPWRK + HH976 SEQ ID NO 979 KFPWWPWRR + HH977 SEQ ID NO 980KKPWWPWRR + HH978 SEQ ID NO 981 KWRWWPWRR ++ HH979 SEQ ID NO 982KWPKWPWRR + HH980 SEQ ID NO 983 KWPWKPWRR + HH981 SEQ ID NO 984KWPWWKWRR ++ HH982 SEQ ID NO 985 KWPWWPKRR + HH983 SEQ ID NO 986KWPWWPWRR +/++ HH984 SEQ ID NO 987 KFRWWPWRR ++ HH985 SEQ ID NO 988KFRWWKWRR ++ HH986 SEQ ID NO 989 KWRWWKKRR ++ HH987 SEQ ID NO 990KKKWWKWRR ++ HH988 SEQ ID NO 991 KFHWWIWRK ++ HH989 SEQ ID NO 992KFHWWKWRK ++ HH990 SEQ ID NO 993 KFKWWKYRK ++ HH991 SEQ ID NO 994KFKFFKYRK + HH992 SEQ ID NO 995 KFKFFKFRK + HH993 SEQ ID NO 996PWWPWRR + HH994 SEQ ID NO 997 KWWPWRR + HH995 SEQ ID NO 998 PWWKWRR +/++HH996 SEQ ID NO 999 RWWPWRR + HH997 SEQ ID NO 1000 PKWPWRR − HH998SEQ ID NO 1001 PWKPWRR − HH999 SEQ ID NO 1002 PWWKWRR + HH1000SEQ ID NO 1003 PWWPKRR − HH1001 SEQ ID NO 1004 PWWPWRK − HH1002SEQ ID NO 1005 RWWKWRR ++ HH1003 SEQ ID NO 1006 RWWKWRK +/++ HH1004SEQ ID NO 1007 RFWKWRR + HH1005 SEQ ID NO 1008 RWWIKRR +/++ HH1006SEQ ID NO 1009 RWWIYRR + HH1007 SEQ ID NO 1010 RFFKFRR − HH1008SEQ ID NO 1011 KWWKWKK + HH1009 SEQ ID NO 1012 KFFKFKK − *Antimicrobialactivity against P. aeruginosa strain H1001 was determined after 4 hoursincubation time with the peptide using luminescence as an indicator(method described above). The antimicrobial activity was ranked usingthe following symbols, − for minimal or no activity, + for weakactivity, +/++ for intermediate activity, ++ strong activity.

Example 4

Development of Semi-Random Peptide Libraries with Enriched AntimicrobialActivities

Semi-random peptide libraries are a simple, powerful tool to createnovel peptide sequences. These novel peptides can be screened for thedesired biological activity. This approach is independent of knowledgeof naturally occurring peptides, and can create sequences from the fullsequence space of all possible peptides. In our first attempts wecreated 200 random 9 amino acid (9-mer) peptides de novo [sequences notincluded with this patent as they are inactive]. For this peptide setcysteine was excluded to avoid the potential for formation of peptidedimers. All peptides were synthesized on cellulose and tested for theirability to kill P. aeruginaosa, using the lux assay and luminescentstrain H1001. After 4 hours incubation time of H1001 with the peptidesno antimicrobial activity was detected (FIG. 4).

Thus a totally random peptide library resulted in essentilly no activeantimicrobial peptides, and this demonstrates that it is not feasible toscreen thousands of random peptides to find a few with antimicrobialactivity. To improve the chances of finding active antimicrobialpeptides, the information gained from previous Bac2A peptide libraries(Hilpert, K., M. R. Elliott, R. Volkmer-Engert, P. Henklein, O. Donini,Q. Zhou, D. F. H. Winkler and R. E. W. Hancock. 2006. Sequencerequirements and a novel optimization strategy for short antimicrobialpeptides. Chem Biol. 13:1101-1107) was used to design new parametersrelated to input amino acid composition to create a semi-randomapproach. Thus instead of using the same occurrence for each amino acid,the occurrence was changed for certain amino acids, according to theiroccurrence in peptides with good activity, as presented in FIG. 5.

Using these new occurrence settings 943 peptide were semi-randomlydesigned and synthesised on cellulose. Twenty eight percent of peptidesdemonstrated similar activity to the control while 2% were more active,and 0.3% demonstrated superior activity. These assessments were repeatedwith 152 of these peptides from the semi-random peptide library and 96%of the activities could be confirmed. Further MIC studies with a randomselection of peptides indicated that the active peptides within thesemi-random library are suitable to use as lead structures for drugdesign.

To further improve the library design, the different activity groupsfound within the first semi-random library were compared with thesetting used to design the first semi random library. The comparison ispresented in FIG. 5. The more active group showed lower usage of theamino acids A, D, E, G, H, M, N, P, Q, S and T compared to the librarysettings. On the other hand the amino acids I, R, V and W were used moreoften compared to the libray settings. Using this information, a secondgeneration semi-random peptide library was designed. The new settingsare presented as a comparison between the first and second librarysettings in FIG. 6.

By using these settings 500 new peptides were designed and synthesizedon cellulose (HH469-HH969), and their antimicrobial activities weretested against H1001. The result of this screen is given in FIG. 7.

Thus the chances of finding an antimicrobial peptide, with activityagainst P. aeruginosa that was comparable to Bac2A or better, using thissecond generation library setting, was greater than 50%. The libraryfeatures could most probably still be optimized since a comparison ofthe amino acid occurrences in the different peptide classes still showedthat there was room for improvement. Thus we used the settings of thesecond generation library adopted combined with a QSAR approach thatutilized our peptide libraries as training sets for the QSAR analysisanddesigned 100,000 peptides.

Example 5

QSAR Analysis as a Route to Predicting New Peptides

The method of Artificial Neural Networks represents one of the mostbroadly used machine-learning techniques that utilize basic principlesof brain organization and memory mechanisms. The structure of a NeuralNetwork mimics three main components of a neural cell and consists of aninput layer where information is entered, one or more hidden layerswhere signals are conducted and processed, and an output layer where theresult of the calculation ends up. Such data flow resembles the passageof an electric signal between neural cells. In short, a dendrite body ofa cell receives multiple input signals from other neurons, and dependingon the intensity of the accumulated input, the activation signal can bepassed to the axon and, hence, along the downstream connections. Justlike a complex biological network of connected neurons, the ArtificialNeural Networks model can learn by example. During the learning phase,it defines the relationship between n input variables Input_nodeij and aknown dependent value Output_nodei by recursive adjustments of theweights attributes wij assigned to each network node. In particular, aset of inputs multiplied by each neuron's weights are summed up for eachof m hidden node:

${Hidden\_ node}_{i} = {\tanh\lbrack {\sum\limits_{i = 1}^{n}\;( {{{Input\_ node}_{i}*w_{ij}} + {{const}_{0}*w_{0}}} )} \rbrack}$Then, the transformed sums for the hidden units are multiplied by theoutput weights:

${Output\_ node} = {\sum\limits_{i = 1}^{m}\;( {{{Hidden\_ node}_{i}*w_{ij}} + {{const}_{0}*w_{0}}} )}$where they are summed a final time and transformed with the learningfunction

$\frac{1}{1 + e^{- x}}$that resembles a sigmoid electric potential occurring between a neuronand dendrite cell. With a trained network, the independent arguments(QSAR descriptors) of an unknown entry (untested peptide) can be passedthough the input nodes and transformed through the pre-defined networkconnections into the output signal (predicted activity). The outputvalues can then be interpreted as active of inactive prediction by itsrespective proximity to 1.0 or 0.0 thresholds.

The common and ‘inductive’ QSAR descriptors described in Table 6 wereused.

TABLE 6 ‘Inductive’ and conventional molecular descriptors utilized inthe QSAR modeling of antimicrobial activity of short cationic peptides.QSAR parameter Description Electronegativity-based EO_EqualizedIteratively equalized electronegativity of a molecule Average_EO_PosArithmetic mean of electronegativities of atoms with positive partialcharge Average_EO_Neg Arithmetic mean of electronegativities of atomswith negative partial charge Hardness-based Sum_Hardness Sum ofhardnesses of atoms of a molecule Sum_Neg_Hardness Sum of hardnesses ofatoms with negative partial charge Average_Hardness Arithmetic mean ofhardnesses of all atoms of a molecule Average_Pos_Hardness Arithmeticmean of hardnesses of atoms with positive partial chargeAverage_Neg_Hardness Arithmetic mean of hardnesses of atoms withnegative partial charge Smallest_Pos_Hardness Smallest atomic hardnessamong values for positively charged atoms Smallest_Neg_Hardness Smallestatomic hardness among values for negatively charged atomsLargest_Pos_Hardness Largest atomic hardness among values for positivelycharged atoms Largest_Neg_Hardness Largest atomic hardness among valuesfor negatively charged atoms Hardness_of_Most_Pos Atomic hardness of anatom with the most positive charge Hardness_of_Most_Neg Atomic hardnessof an atom with the most negative charge Softness basedTotal_Neg_Softness Sum of softnesses of atoms with negative partialcharge Average_Neg_Softness Arithmetic mean of softnesses of atoms withnegative partial charge Charge-based Average_Pos_Charge Arithmetic meanof positive partial charges on atoms of a molecule Average_Neg_ChargeArithmetic mean of negative partial charges on atoms of a moleculeDescriptors based on inductive substituent constants Total_Sigma_mol_iSum of inductive parameters sigma (molecule→atom) for all atoms within amolecule Most_Pos_Sigma_mol_i Largest positive group inductive parametersigma (molecule→atom) for atoms in a molecule Most_Neg_Sigma_mol_iLargest (by absolute value) negative group inductive parameter sigma(molecule→atom) for atoms in a molecule Sum_Pos_Sigma_mol_i Sum of allpositive group inductive parameters sigma (molecule→atom) within amolecule Sum_Neg_Sigma_mol_i Sum of all negative group inductiveparameters sigma (molecule→atom) within a molecule Descriptors based onsteric substituent constants Smallest_Rs_mol_i Smallest value of groupsteric influence Rs(molecule→atom) in a molecule Largest_Rs_i_molLargest value of atomic steric influence Rs(atom→molecule) in a moleculeMost_Neg_Rs_mol_i Steric influence Rs(molecule→atom) ON the mostnegatively charged atom in a molecule Most_Neg_Rs_i_mol Steric influenceRs(atom→molecule) OF the most negatively charged atom to the rest of amolecule Conventional QSAR descriptors implemented by the MolecularOperational Environment (MOE) v. 2006.05 software, Chemical ComputationGroup Inc., Montreal, Canada. a_acc Number of hydrogen bond acceptoratoms a_don Number of hydrogen bond donor atoms ASA Water accessiblesurface area ASA_H Water accessible surface area of all hydrophobicatoms ASA_P Water accessible surface area of all polar atoms ASA− Wateraccessible surface area of all atoms with negative partial charge ASA+Water accessible surface area of all atoms with positive partial chargeFCharge Total charge of the molecule logP(o/w) Log of the octanol/waterpartition coefficient logS Log of the aqueous solubility PC− Totalnegative partial charge PC+ Total positive partial charge RPC+ Relativepositive partial charge vdw_area van der Waals surface area calculatedusing a connection table approximation vsa_acc Approximation to the sumof VDW surface areas of pure hydrogen bond acceptors vsa_acidApproximation to the sum of VDW surface areas of acidic atoms vsa_hydApproximation to the sum of VDW surface areas of basic atoms WeightMolecular weight

The conventional QSAR descriptors were calculated for the training setand external set compounds using the default setting of the MOE package,while the ‘inductive’ parameters have been calculated by customized SVLscripts (a specialized language of the MOE) using the fundamentalequations found in FIG. 8) for steric effect parameters, parameters ofinductive influence, ‘inductive’ partial charge, group ‘inductive’electronegativity and ‘inductive’ analogues of local and global chemicalhardness and softness. The linear character of these equations made theinductive descriptors in FIG. 8 readily computable and suitable forsizable databases and positions them as appropriate parameters forlarge-scale QSAR models.

The interatomic distances were calculated for all evaluated peptidesusing their three-dimensional structures optimized with MMFF94force-field. The atomic types have been assigned according to the name,valent state and a formal charge of atoms as it is defined within theMOE.

The QSAR descriptors used in the study have been normalized into therange [0.0÷1.0] and the non-overlapping training and testing sets havebeen randomly drawn by the customized Java scripts. The training andtesting of the neural networks has been conducted using the StuttgartNeural Network Simulator. The training was performed through thefeed-forward back-propagation algorithm with the weight decay andpattern shuffling. The values of initial rates were randomly assigned ina range [0.0÷1.0], the learning rate has been set to 0.8 with thethreshold 0.10. The external set of 100,000 peptide candidates designedusing the second generation library parameters described in FIG. 6 wascreated using customized SVL scripts.

Example 6

Prediction of Novel Peptides

To relate QSAR descriptors to known antimicrobial activity of previouslystudied peptides, as described in Example 5, the method of ArtificialNeural Networks, one of the most effective pattern recognitiontechniques that is ranked very highly among machine learning approaches,was employed. Two training datasets of 943 peptides (Hilpert K, and R EW Hancock, unpublished) and 500 peptides (FIG. 7) were dealt withseparately, since they were assayed at different times under slightlydifferent conditions, and in combined collection of 933+500=1433substances and consequently trained three independent Neural Networkmodels respectively based on the training sets ‘A’, ‘B’ and ‘A+B’.

Within each of those training sets, an output value of 1.0 was assignedfor the most active (top 5%) of the peptides and 0.0 values were usedfor all others. With this, multiple training runs of the Neural Networkswere carried out, while changing the number of their hidden nodes. Asthe result, it was established that the most optimal performance by theNetwork-based solutions was achieved with 10 nodes in the hidden layer.Using this optimized 44-10-1 configuration of the Neural Network QSARsolutions were trained using 10-folds cross-validation technique. Inparticular, for each training set ‘A’, ‘B’ and ‘A+B’, 10 independentmodels were created, each derived from a 90% portion of the trainingset. Every solution was then applied to the remaining 10% of data thathad been excluded from the training process. Thus, for every peptide inthe training sets ‘A’, ‘B’ and ‘A+B’ 10 predictions were computed andthese were further averaged as arithmetic means. The averaged outputswere then interpreted as active/inactive predictions by applying thepreviously utilized top 5% (most active peptides) criterion. Finally,the predicted outcomes were compared with experimental peptideactivities (also separated into the top 5% vs. remaining 95%) to produceconfusion matrices. The resulting parameters of Specificity,Sensitivity, Accuracy and the Positive Predictive Value observed whendelineating the top 5, 10 or 25% of peptides as the “most active” arepresented in Table 7.

TABLE 7 Parameters characterizing the ability of the Neural Networks torecognize the most active peptides in training sets A, B and ‘A + B’containing known antimicrobial peptides. Top % as Positive Training“most Predictive set actives” Accuracy Specificity Sensitivity Value A 5% 0.96 0.98 0.62 0.58 10% 0.93 0.94 0.76 0.39 25% 0.78 0.78 0.85 0.17B  5% 0.94 0.97 0.33 0.30 10% 0.88 0.90 0.33 0.12 25% 0.77 0.77 0.800.12 A + B  5% 0.95 0.97 0.47 0.47 10% 0.91 0.92 0.54 0.27 25% 0.76 0.770.66 0.13

In addition, all three developed QSAR models were assessed using theReceiver Operating Characteristics curves (plotting average truepositive rates as a function of average false positive rates. Thecomputed ‘area under the curve’ values of Training set A=0.87, B=0.83and A+B=0.80 confirmed the accuracy of these QSAR models anddemonstrated that the selected set of 44 QSAR descriptors can adequatelycapture structural properties of peptides that are relevant for theirantibacterial activities.

In silico interrogation of designed peptide libraries. To utilize thedeveloped QSAR solutions further, 100,000 virtual variants of 9-aminoacid long peptides were created using the favorable proportions of aminoacids ustilized for the second generation library as described in FIG.6.

At the next step we calculated 44 QSAR parameters for each virtualpeptide and scored all 100,000 of them with 30 neural network-based QSARsolutions created with the training sets ‘A’, ‘B’ and ‘A+B’ anddescribed in the previous section. Thus, for every hypothetical peptidewe produced 30 independent network outputs representing hypotheticalantimicrobial potentials, but instead of averaging, they were subjectedto a binary voting system. In particular, after sorting 30 sets ofpredicted activities, the cumulative votes were computed for ˜100,000peptides, whereby each peptide would receive a vote of 1 for every top5% ranking (thus, the maximal possible value was set to 30). Inaddition, the cumulative ranks of peptides were also computed. Theactivity prediction for the 100,000 peptides is summarized in Table 7(see appendix) and assorted according to quartiles (Most activepredicted quartile to least active).

To test the accuracy of predictions, fifty peptides were taken from theboundaries of each quartile (total of 200 peptides) and resynthesized oncellulose arrays and tested for antimicrobial activity using theluminescence assay described above, see Table 7.

Results were as follows:

-   -   For the first 50 (representing the first quartile), 47 of them        (94%) were more active than the control Bac2A, with only 3        peptide being as active as the control.    -   For the second 50 (representing the second quartile) 32 of them        (64%) were more active than the control, while 17 peptides were        similar or worse than control and 1 peptide was inactive.    -   For the third 50 (representing the third quartile) Only 8 (16%)        similar to or better than the control, 38 were worse than the        control and 4 were inactive    -   For the bottom 50 (representing the fourth quartile with lowest        predicted activity) 44 were worse than control and 6 were        inactive.

Thus it is quite clear that the QSAR-derived model was very accurate inpredicting peptides with excellent antimicrobial activity. See FIG. 20.

TABLE 8 Selected peptides from the 100,000 peptide set. MeasuredPredicted activity activity Name Sequence (in Quartiles) (IC₅₀) HHC1RWRWKRWWW 1 0.25 HHC2 RWRRWKWWW 1 0.40 HHC3 RWWRWRKWW 1 0.28 HHC4RWRRKWWWW 1 0.39 HHC5 RWRWWKRWY 1 0.20 HHC6 RRKRWWWWW 1 0.43 HHC7RWRIKRWWW 1 0.12 HHC8 KIWWWWRKR 1 0.13 HHC9 RWRRWKWWL 1 0.078 HHC10KRWWKWIRW 1 0.037 HHC11 KRWWWWWKR 1 0.22 HHC12 IRWWKRWWR 1 0.21 HHC13IKRWWRWWR 1 0.23 HHC14 RRKWWWRWW 1 0.27 HHC15 RKWWRWWRW 1 0.31 HHC16KRWWWWRFR 1 0.24 HHC17 IKRWWWRRW 1 0.22 HHC18 KRWWWVWKR 1 0.36 HHC19KWRRWKRWW 1 0.15 HHC20 WRWWKIWKR 1 0.14 HHC21 WRWRWWKRW 1 0.28 HHC22WKRWKWWKR 1 0.25 HHC23 RIKRWWWWR 1 0.31 HHC24 IWKRWWRRW 1 0.24 HHC25KWWKIWWKR 1 0.20 HHC26 RKRWLWRWW 1 0.25 HHC27 KRWRWWRWW 1 0.28 HHC28KKRWLWWWR 1 0.30 HHC29 RWWRKWWIR 1 0.24 HHC30 KWWRWWRKW 1 0.20 HHC31KRWWIRWWR 1 0.21 HHC32 KIWWWWRRR 1 0.21 HHC33 RRRKWWIWW 1 0.18 HHC34RRRWWWWWW 1 1.8 HHC35 RWWIRKWWR 1 0.21 HHC36 KRWWKWWRR 1 0.13 HHC37KRWWRKWWR 1 0.15 HHC38 RRIWRWWWW 1 0.68 HHC39 IRRRKWWWW 1 0.21 HHC40KRKIWWWIR 1 0.28 HHC41 RKIWWWRIR 1 0.59 HHC42 KRWWIWRIR 1 0.35 HHC43RWFRWWKRW 1 0.26 HHC44 WRWWWKKWR 1 0.19 HHC45 WKRWWKKWR 1 0.20 HHC46WKRWRWIRW 1 0.28 HHC47 WRWWKWWRR 1 0.23 HHC48 WKKWWKRRW 1 0.19 HHC49WRWYWWKKR 1 0.22 HHC50 WRRWWKWWR 1 0.23 HHC51 IRMWVKRWR 2 0.61 HHC52RIWYWYKRW 2 0.36 HHC53 FRRWWKWFK 2 0.12 HHC54 RVRWWKKRW 2 0.27 HHC55RLKKVRWWW 2 0.34 HHC56 RWWLKIRKW 2 0.18 HHC57 LRWWWIKRI 2 0.33 HHC58TRKVWWWRW 2 0.76 HHC59 KRFWIWFWR 2 3.0 HHC60 KKRWVWVIR 2 0.35 HHC61KRWVWYRYW 2 0.54 HHC62 IRKWRRWWK 2 0.41 HHC63 RHWKTWWKR 2 0.95 HHC64RRFKKWYWY 2 0.26 HHC65 RIKVIWWWR 2 0.51 HHC66 RKRLKWWIY 2 0.18 HHC67LVFRKYWKR 2 0.99 HHC68 RRRWWWIIV 2 0.85 HHC69 KKRWVWIRY 2 0.22 HHC70RWRIKFKRW 2 0.26 HHC71 KWKIFRRWW 2 0.16 HHC72 IWKRWRKRL 2 0.33 HHC73RRRKWWIWG 2 0.57 HHC74 RWLVLRKRW 2 0.53 HHC75 RKWIWRWFL 2 0.15 HHC76KRRRIWWWK 2 0.40 HHC77 IWWKWRRWV 2 0.29 HHC78 LRWRWWKIK 2 0.26 HHC79RWKMWWRWV 2 0.24 HHC80 VKRYYWRWR 2 1.2 HHC81 RWYRKRWSW 2 0.70 HHC82KRKLIRWWW 2 0.23 HHC83 RWRWWIKII 2 0.46 HHC84 KFRKRVWWW 2 0.30 HHC85IWIWRKLRW 2 0.46 HHC86 LRFILWWKR 2 0.88 HHC87 RVWFKRRWW 2 0.26 HHC88RRWFVKWWY 2 0.52 HHC89 KWWLVWKRK 2 0.23 HHC90 RWILWWWRI 2 25 HHC91KRWLTWRFR 2 0.54 HHC92 RKWRWRWLK 2 0.31 HHC93 IRRRWWWIV 2 0.23 HHC94IKWWWRMRI 2 0.39 HHC95 RWKIFIRWW 2 1.8 HHC96 IRQWWRRWW 2 0.50 HHC97RRRKTWYWW 2 0.32 HHC98 RRWWHLWRK 2 0.38 HHC99 RRWWMRWWV 2 0.33 HHC100RRFKFIRWW 2 0.24 HHC101 INRKRRLRW 3 4.2 HHC102 RRMKKLRRK 3 4.2 HHC103RKVRWKIRV 3 0.32 HHC104 VRIVRVRIR 3 2.2 HHC105 IKRVKRRKR 3 2.9 HHC106RVKTWRVRT 3 5.7 HHC107 RVFVKIRMK 3 0.72 HHC108 IRGRIIFWV 3 0.44 HHC109ATWIWVFRR 3 4.9 HHC110 KKSKQLWKR 3 3.2 HHC111 MINRVRLRW 3 2.8 HHC112GGIRRLRWY 3 1.2 HHC113 RLVHWIRRV 3 2.6 HHC114 AWKIKKGRI 3 3.6 HHC115FVVMKRIVW 3 5.4 HHC116 GIKWRSRRW 3 1.1 HHC117 RWMVSKIWY 3 25 HHC118IVVRVWVVR 3 3.5 HHC119 RWIGVIIKY 3 2.2 HHC120 WIRKRSRIF 3 3.4 HHC121GWKILRKRK 3 2.7 HHC122 YQRLFVRIR 3 25 HHC123 AVWKFVKRV 3 8.2 HHC124IRKKRRRWT 3 6.6 HHC125 ILRVISKRR 3 25 HHC126 AWRFKNIRK 3 9.2 HHC127HYKFQRWIK 3 2.8 HHC128 RRIRRVRWG 3 8.2 HHC129 VLVKKRRRR 3 12 HHC130RWRGIVHIR 3 4.9 HHC131 WRNRKVVWR 3 6.8 HHC132 KFWWWNYLK 3 1.8 HHC133KRIMKLKMR 3 6.5 HHC134 IRRRKKRIK 3 6.4 HHC135 RKWMGRFLM 3 4.4 HHC136RRVQRGKWW 3 6.3 HHC137 WHGVRWWKW 3 2.5 HHC138 WVRFVYRYW 3 2.1 HHC139RKRTKVTWI 3 5.1 HHC140 IRRIVRRKI 3 11.1 HHC141 KIRRKVRWG 3 10.6 HHC142AIRRWRIRK 3 4.6 HHC143 WRFKVLRQR 3 7.1 HHC144 RSGKKRWRR 3 6.5 HHC145FMWVYRYKK 3 1.5 HHC146 RGKYIRWRK 3 3.8 HHC147 WVKVWKYTW 3 5.6 HHC148VVLKIVRRF 3 25 HHC149 GKFYKVWVR 3 1.2 HHC150 SWYRTRKRV 3 6.7 HHC151KNRGRWFSH 4 9.8 HHC152 AFRGSRHRM 4 11 HHC153 GRNGWYRIN 4 11 HHC154AGGMRKRTR 4 25 HHC155 ATRKGYSKF 4 25 HHC156 SSGVRWSWR 4 8.2 HHC157RVWRNGYSR 4 10 HHC158 WGRTRWSSR 4 9.6 HHC159 GKRVWGRGR 4 8.2 HHC160SFNWKRSGK 4 25 HHC161 WGRGGWTNR 4 25 HHC162 ANRWGRGIR 4 11 HHC163WGGHKRRGW 4 6.2 HHC164 WHGGQKWRK 4 8.5 HHC165 FVWQKGTNR 4 11 HHC166HGVWGNRKR 4 7.9 HHC167 TRGWSLGTR 4 12 HHC168 GRRVMNQKR 4 9.8 HHC169RNKFGGNWR 4 25 HHC170 GVRVQRNSK 4 25 HHC171 NQKWSGRRR 4 8.0 HHC172RQNGVWRVF 4 8.3 HHC173 GRMRLWNGR 4 7.9 HHC174 WHYRSQVGR 4 6.6 HHC175GWNTMGRRW 4 6.3 HHC176 RRMGNGGFR 4 8.7 HHC177 SKNVRTWRQ 4 7.6 HHC178ARGRWINGR 4 7.2 HHC179 GSRRSVWVF 4 2.3 HHC180 WSQNVRTRI 4 5.7 HHC181GMRRWRGKN 4 6.0 HHC182 RGRTSNWKM 4 7.1 HHC183 GRRWGMGVR 4 7.7 HHC184WGKRRGWNT 4 7.9 HHC185 AMLGGRQWR 4 6.7 HHC186 QRNKGLRHH 4 8.8 HHC187ARGKSIKNR 4 8.3 HHC188 NRRNGQMRR 4 8.4 HHC189 RGRRQIGKF 4 8.5 HHC190ASKRVGVRN 4 8.2 HHC191 GRIGGKNVR 4 9.1 HHC192 NKTGYRWRN 4 8.3 HHC193VSGNWRGSR 4 8.5 HHC194 GWGGKRRNF 4 7.3 HHC195 KNNRRWQGR 4 6.4 HHC196GRTMGNGRW 4 6.9 HHC197 GRQISWGRT 4 8.0 HHC198 GGRGTRWHG 4 8.6 HHC199GVRSWSQRT 4 8.5 HHC200 GSRRFGWNR 4 8.1 The predicted activities aregiven in activity quartiles, where the most active predicted peptidequartile (top 25,000 peptides) is Quartile 1, Quartiles 2 and 3 arepredicted to be successively less active and the least active ispredicted to be Quartile 4. The antimicrobial activity of these peptideswas determined by the luminescence assay. The activity was determined bygraphing the luminescence values as a function of peptide concentration.The highest peptide concentration was set to 1. As a consequence, thedetermined IC₅₀ values, rounded to 2 significant figures, are relative(Hilpert, K., and R. E. W. Hancock, Use of luminescent bacteria forrapid screening and characterization of short cationic antimicrobialpeptides synthesized on cellulose using peptide array technology, NatureProtocols, 2007, vol. 2, pp. 1652-1660).

To further evaluate the accuracy of the developed structure-activitymodels 25 peptide candidates (Table 9) were selected at random from theabove-described 200 peptides and representing the entire range ofpredicted activities. Thus five to eight sequences were selected fromeach quartile of the 100,000 predicted peptides sorted by theircumulative votes and ranking. Thus, the collection of 25 selectedpeptides was expected to contain high-, median-, low- and completelyinactive entries (roughly corresponding to the quartiles).

The selected peptides were synthesized and assayed against several majorantibiotic-resistant pathogens. Initially, the peptides were screenedagainst the laboratory strain of P. aeruginosa PAO1 to compare theresults with the training data. It was confirmed that peptide candidatesselected from the ‘fourth quartile’ did not posses any antimicrobialactivity, as had been forecasted by the QSAR (HHC-152, HHC-183, HHC-186,HHC-189, and HHC-190). The antibacterial activity in the form of minimalinhibitory concentration (MIC) of the other studied peptides wasassessed in greater depth (Table 9) against many highly antibioticresistanct pathogens.

TABLE 9 MIC activity values for the QSAR-designed peptides. MIC (μM)Peptide Sequence PA A B C D E F G Bac2A RLARIVVIRVAR 35 48 192 95 12 324 24 HHC-8 KIWWWWRKR 5 6 47 24 5.9 3 94 6 HHC-9 RWRRWKWWL 37 3 12 120.3 0.7 6 3 HHC-10 KRWWKWIRW 1.4 0.8 6 1.5 0.8 0.4 3 1.5 HHC-20WRWWKIWKR 5 6 24 24 1.5 0.8 12 6 HHC-36 KRWWKWWRR 4 0.7 5.7 1.4 0.3 1.411 3 HHC-45 WKRWWKKWR 7 23 46 46 6 1.4 93 3 HHC-48 WKKWWKRRW 7 23 46 466 1.4 23 3 HHC-53 FRRWWKWFK 4.2 1.5 12 3.0 1.5 0.8 24 6 HHC-57 LRWWWIKRI12 13 50 25 6 3 50 13 HHC-66 RKRLKWWIY 7 25 50 50 6 3 13 6 HHC-69KKRWVWIRY 8 25 51 25 3 1.6 25 13 HHC-71 KWKIFRRWW 6 12 24 24 3 1.5 6 12HHC-75 RKWIWRWFL 5 6 12 3 1.5 1.5 3 1.5 HHC-77 IWWKWRRWV 10 6 48 12 61.5 6 6 HHC-100 RRFKFIRWW 9 6 24 49 3 0.8 12 12 HHC-123 AVWKFVKRV 360240 >240 240 120 60 >240 120 HHC-126 AWRFKNIRK 376 >223 >223 >223111 >223 >223 223 HHC-133 KRIMKLKMR269 >226 >226 >226 >226 >226 >226 >226 HHC-142 AIRRWRIRK184 >217 >217 >217 217 108 108 108 HHC-148 VVLKIVRRF 1104 >241 >241 >241241 60 241 241 HHC-152 AFRGSRHRM 506 NT NT NT NT NT NT NT HHC-183GRRWGMGVR 360 NT NT NT NT NT NT NT HHC-186 QRNKGLRHH 381 NT NT NT NT NTNT NT HHC-189 RGRRQIGKF 379 NT NT NT NT NT NT NT HHC-190 ASKRVGVRN 413NT NT NT NT NT NT NT MIC (μM) Peptide H I J K L M N O Bac2A 192 24 24 1248 12 3 48 HHC-8 47 6 6 24 94 6 1.5 94 HHC-9 11 3 3 23 92 6 1.4 92HHC-10 6 3 1.5 12 99 3 1.5 49 HHC-20 24 3 3 24 94 6 1.5 94 HHC-36 22 31.4 43 >174 11 1.3 174 HHC-45 46 68 6 93 >186 23 6 >186 HHC-48 46 1.4 3993 >186 12 6 >186 HHC-53 24 1.5 3 24 195 6 6 97 HHC-57 50 6 63 13 50 61.5 25 HHC-66 50 6 6 50 >202 13 3 202 HHC-69 51 13 13 25 102 6 6 102HHC-71 97 3 3 24 97 24 6 97 HHC-75 3.1 31 3 6 24 3 3 24 HHC-77 12 3 3 2448 6 3 48 HHC-100 49 6 6 12 98 6 6 49 HHC-123 >240 240 240 >240 >240 120120 >240 HHC-126 >223 223 >223 >223 >223 >223 223 >223HHC-133 >226 >226 >226 >226 >226 >226 >226 >226 HHC-142 >217 5454 >217 >217 108 14 >217 HHC-148 >241 241 241 241 >241 241 60 >241HHC-152 NT NT NT NT NT NT NT NT HHC-183 NT NT NT NT NT NT NT NT HHC-186NT NT NT NT NT NT NT NT HHC-189 NT NT NT NT NT NT NT NT HHC-190 NT NT NTNT NT NT NT NT Column legends: PA01, P. aeruginosa Lab strain; A, P.aeruginosa wild type strain H103; B, C, P. aeruginosa multidrugresistant strains from Brazil strain H9 and H123; D, P. aeruginosamultidrug resistant Liverpool epidemic strains H1031, H1030, and H1027respectively; E, multidrug resistant Pseudomonas maltophilia ATCC13637;F, Extended-Spectrum β-lactamase-producing β-lactam resistant (ESBL)Enterobacter cloacae strain C601. G, ESBL E. coli clinical strain 64771;H, ESBL Klebsiella pneumonia clinical strain 63575; I, S. aureusATCC25923; J, Methicillin resistant S. aureus (MRSA) strain C623; K,Enterococcus faecalis ATCC29212; L, M, VRE Vancomycin resistantEnterococcus faecalis clinical isolates w61950 (VanA) and f43559 (VanB);N, O, VRE Vancomycin resistant Enterococcus faecium clinical isolatesmic80 (VanA) and t62764 (VanB).

All of the peptides in Table 9 had similar physical properties. Theseexperimental results unambiguously demonstrated that the QSAR approachis able to be utilized for accurately forecasting the antimicrobialactivity of de novo designed peptides. Thus, all 7 ‘first-quartile’derivatives demonstrated very significant activity against P. aeruginosaPAO1 with the corresponding MIC parameters ranging from 1.4 μM to 6.8μM. Moreover, 2 out of 7 ‘first-quartile’ peptides (HHC-8, HHC-9,HHC-10, HHC-20, HHC-36, HHC-45, and HHC-48), actually outperformed themost active lead from the training ‘set A’ (with MIC=3.29 μM).Interestingly enough, there were only 6 peptides with MIC<7 μM could befound in the entire ‘set A’. In another pre-designed training ‘set B’only 1 out of 500 entries demonstrated an MIC<6 μM. Importantly,peptides selected from the ‘second quartile’ also demonstratedsubstantial antimicrobial activity in that their MIC values rangedbetween 4 μM and 12 μM (HHC-53, HHC-57, HHC-66, HHC-69, HHC-71, HHC-75,HHC-77, and HHC-100). As predicted by the QSAR, the third quartileselection (HHC-123, HHC-126, HHC-133, HHC-142, HHC-148) did not returnany generally active substances, while the fourth quartile peptides wereall virtually completely inactive.

These results clearly illustrate that the QSAR approach can accuratelypredict the antimicrobial activity of peptides and permit thedevelopment of structure-activity models that create lists of drugcandidates. To illustrate that important observation, we derived medianMICs for P. aeruginosa PAO1 for the training sets A (91 μM) and B (127μM) compared to the corresponding median MICs for the experimentallytested peptides from the 1^(st), 2^(nd), 3^(rd), and 4^(th) quartiles(7, 13, 172 and 379 μM respectively). Thus, these results demonstratedthe superior performance of atom-based QSAR approach compared toconventional peptide design strategies traditionally relying on charge,hydrophobicity and/or amphipathicity properties of isolated aminoacids.To illustrate this notion further, median values of formal charge,hydrophobic fraction and hydrophobic moment were computed for peptidespresent in the training sets A and B as well as for all 100,000predicted candidates populating the quartiles (FIG. 9).

This chart clearly demonstrates that there was very limited variation incharge (Q), hydrophobicity (P) and hydrophobic moments (HM) of peptidesin the 4 activity quartiles, while their antimicrobial activities variedtremendously. Thus adequate modeling of antimicrobial activity ofcationic peptides demands substantially more refined structure-activityapproaches including a detailed, atomic-level of consideration ofmolecular structures, rather than simplistic consideration of the polarand hydrophobic characteristics of constituent aminoacids.

All peptides derived from the 1^(st), 2^(nd), and 3^(rd) quartiles werefurther evaluated for their antibacterial activities against severalhighly antibitoic resistant pathogens (Table 3). These included clinicalisolates of MRSA, ESBL E. cloacae, and multidrug resistant Pseudomonasstrains including Brazilian clinical isolates of P. aeruginosa that areresistant to meropenem, ceftazidime, piperacillin/tazobactam,ciprofloxacin, cefepime and polymyxin B, and the Liverpool EpidemicStrains. All 15 peptides from the 1^(st) and 2^(nd) quartilesdemonstrated significant activity against resistant strains, andeffectively inhibited bacterial growth at low μM concentrations. Somecandidates such as HHC-9, HHC-10, HHC-36 and HHC-75 exhibited 1-10 μMactivity against nearly all tested superbugs. Such results characterizethe developed peptides as excellent antibiotic candidates, providing newmeans for treating most dangerous and severe forms of human infections.

To further confirm this, a mouse model of aggressive bacterialinfection, widely used to assess antibiotic efficacy, was utilized. Micewere treated with 1.6×10¹⁰ CFU IP. Four hours post infection theyreceived a dose of 4 mg/kg peptide IP. The infection was allowed toprogress for 20 more hours, for a total infection time of 24 hours.Control mice injected with just saline demonstrated 100% death; incontrast HHC-10 protected 40% of mice while HHC-36 protected 60% ofmice. A second experiment with administration of 1.4×10¹⁰ IP resulted insignificant reduction in the number of bacteria in the animals givenpeptide (FIG. 10).

To assess possible host toxicity of the developed compounds we alsotested 20 peptides for their hemolytic activity (FIG. 11) demonstratingthat the developed antibiotics do not affect host cells.

Amongst the preferred nine amino acid antimicrobial peptides, a clearpattern of related peptides were found that obviously represented minorsubstitutions, deletions or additions to a base sequence represented bySEQ ID NO: 1022. Thus these peptides have a clear unitary relationship.In the following sequence alignments bolded letters represent aminoacids that are identical or represent conservative substitutions (i.e.,hydrophobic amino acid substitutions A, L, V, W, I, or F; or chargesubstitutions R or K).

HHC-10 KRWWK-WIRW SEQ ID NO: 1022 HHC-36 KRWWK-WWRR SEQ ID NO: 1048HHC-8 KIWWW-W-RKR SEQ ID NO: 1020 HHC-20 WRWWKIWKR SEQ ID NO: 1032HHC-45 WKRWWKKW-R SEQ ID NO: 1057 HHC-48 WKKWWKR-RW SEQ ID NO: 1060

EXAMPLE 7

Anti-Septic Impact on Innate Immunity

It is well known that cationic antimicrobial peptides have the abilityto boost immunity while suppressing septic responses to bacterialpathogen associated molecular pattern molecules like lipopolysaccharideand lipoteichoic acids as well as reducing inflammation and endotoxaemia(Finlay, B. B., and R. E. W. Hancock. 2004. Can innate immunity beenhanced to treat infections? Nature Microbiol. Rev. 2:497-504).

Small 12-mer peptides like Bac2A and 13-mer peptides like indolicidinhave been previously shown in our laboratory to have rather modestanti-endotoxic activity, which can be assessed by measuring the abilityof the peptide to suppress the LPS-stimulated production of TNFα bymacrophages. It is well known for other cationic antimicrobial peptidesthat this corresponds to anti-endotoxic activity in reversing lethalendotoxaemia in animal models (Gough M, Hancock R E W, and Kelly N M.1996. Anti-endotoxic potential of cationic peptide antimicrobials.Infect. Immun. 64, 4922-4927). In contrast LL-37 is known to haveexcellent anti-endotoxic activity in vitro, as assessed by its abilityto suppress the LPS-mediated induction of TNFα in monocytic cells andthis is reflected by its ability to both reduce endotoxin mediated TNFαinduction and lethality in a mouse model (Scott, M. G., D. J. Davidson,M. R. Gold, D. Bowdish, and R. E. W. Hancock. 2002. The humanantimicrobial peptide, LL-37, is a multifunctional modulator of innateimmune responses. J. Immunol. 169:3883-3891). A selection of peptideswere tested and some of these indeed had excellent anti-endotoxicactivity (FIG. 12).

Only three of the peptides showed any evidence of cytotoxicity towardTHP-1 cells, and this was only evident at 100 μg/ml of peptide (Table10). In addition the following peptides were tested for LDH release:1002, 1005, 1012, 1010, 1013, 1018, 1020, 1026, 1028, 1032, 1033, 1035,and 1037. None showed any LDH release even at 200 mg/ml.

TABLE 10 Cytotoxicity of peptides against THP-1 cells tested at 10 and100 μg/ml. Name Cytotoxicity HH1 No cytotoxicity observed HH2 Nocytotoxicity observed HH3 No cytotoxicity observed HH4 No cytotoxicityobserved HH5 50-60% at 100 μg/ml HH6 No cytotoxicity observed HH7 Nocytotoxicity observed HH8 No cytotoxicity observed HH14 No cytotoxicityobserved HH15 25% at 100 μg/ml HH16 80% at 100 μg/ml HH17 Nocytotoxicity observed

LPS from P. aeruginosa strain H103 was highly purified free of proteinsand lipids using the Darveau-Hancock method. Briefly, P. aeruginosa wasgrown overnight in LB broth at 37° C. Cells were collected and washedand the isolated LPS pellets were extracted with a 2:1chloroform:methanol solution to remove contaminating lipids. PurifiedLPS samples were quantitated using an assay for the specific sugar2-keto-3-deoxyoctosonic acid (KDO assay) and then resuspended inendotoxin-free water (Sigma-Aldrich).

Human monocytic cells, THP-1, were obtained from American type culturecollection, ATCC® (TIB-202) and were grown in suspension in RPMI-1640media (Gibco®, Invitrogen™ Life technologies, Burlington, ON),supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 2mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen LifeTechnologies). Cultures were maintained at 37° C. in a humidified 5%(v/v) CO₂ incubator up to a maximum of six passages. THP-1 cells at adensity of 1×10⁶ cells/ml were treated with 0.3 μg/ml phorbol12-myristate 13-acetate (PMA; Sigma-Aldrich Canada, Oakville ON) for 24hours, inducing plastic-adherent cells that were further rested incomplete RPMI-1640 medium for an additional 24 hours prior tostimulation with various treatments including P. aeruginosa LPS (10ng/ml) with or without peptides for 24 hours after which supernatantswere collected and TNFα assessed by ELISA.

THP-1 cells were stimulated with LPS (10 ng/ml) with or without peptide(10 or 100 μg/ml) for 4 hours as indicated in the results section.Following incubation of the cells under various treatment regimens, thetissue culture supernatants were centrifuged at 1000×g for 5 min, thenat 10,000×g for 2 min to obtain cell-free samples. Supernatants werealiquoted and then stored at −20° C. prior to assay for variouscytokines. TNFα secretion was detected with a capture ELISA (eBioscienceand BioSource International Inc., CA, USA respectively).

The data in FIG. 12 demonstrated that LPS as expected induced largelevels of TNFα. This was strongly suppressed by the control peptideLL-37, as well as by the novel peptides HH2, HH3, HH6, HH8, HH15 andHH16. In addition several of the remaining peptides, including HH1, HH5,and HH17 caused no significant increase in TNFα production.

Anti-endotoxin effects of peptides derived from indolicidin. Somesmaller peptides, like indolicidin (Bowdish D M, Davidson D J, Scott MG, Hancock R E W. Immunomodulatory activities of small host defensepeptides. Antimicrobial Agents Chemotherapy 49:1727-32, 2005), are knownto be able to inhibit the production of proinflammatory cytokines likeTNFα in repsonse to endotoxin. Therefore a variety of peptides derivedfrom indolicidin were tested for their ability to inhibit TNFα responsesafter challenge with P. aeruginosa LPS. The results are presented inFIG. 13. Basically we were able to demonstrate that the followingpeptides HH63, HH111, HH117, HH235, HH973, HH1010 and HH1011 wereslightly better or equivalent than indolicidin.

EXAMPLE 8

Enhancement of Innate Immunity

The natural human peptide LL-37 is able to protect against bacterialinfections despite having no antimicrobial activity under physiologicalconditions (Bowdish, D. M. E., D. J. Davidson, Y. E. Lau, K. Lee, M. G.Scott, and R. E. W. Hancock. 2005. Impact of LL-37 on anti-infectiveimmunity. J. Leukocyte Biol. 77:451-459). It appears to manifest thisactivity due to its ability to induce the production of certainchemokines which are able to recruit subsets of cells of innate immunityto infected tissues. Therefore we tested if the novel peptides describedhere had the ability to induce chemokine production in human peripheralblood mononuclear cells.

Venous blood (20 ml) from healthy volunteers was collected inVacutainer® collection tubes containing sodium heparin as ananticoagulant (Becton Dickinson, Mississauga, ON) in accordance with UBCethical approval and guidelines. Blood was diluted 1:1 with completeRPMI 1640 medium and separated by centrifugation over a Ficoll-Paque®Plus (Amersham Biosciences, Piscataway, N.J., USA) density gradient.White blood cells were isolated from the buffy coat, washed twice inRPMI 1640 complete medium, and the number of peripheral bloodmononuclear cells (PBMC) was determined by trypan blue exclusion. PBMC(5×10⁵) were seeded into 12-well tissue culture dishes (Falcon; BectonDickinson) at 0.75 to 1×10⁶ cells/ml at 37° C. in 5% CO₂. The aboveconditions were chosen to mimic conditions for circulating bloodmonocytes entering tissues at the site of infection via extravasation.

Following incubation of the cells under various treatment regimens, thetissue culture supernatants were centrifuged at 1000×g for 5 min, thenat 10,000×g for 2 min to obtain cell-free samples. Supernatants werealiquoted and then stored at −20° C. prior to assay for variouschemokines by capture ELISA (eBioscience and BioSource InternationalInc., CA, USA respectively)

As shown in FIG. 14, most of the peptides stimulated the expression ofthe neutrophils chemokine IL8 even at the lowest peptide concentrationutilized (20 μg/ml). Peptides HH2, HH4, HH7, HH8, HH13, HH7, HH14, andHH18 appeared to have the strongest abilities to induce this chemokine.

The monocyte chemokine MCP1 (FIG. 15) was also induced by a subset ofthe peptides including especially, HH1, HH2, HH4, HH7, HH8, HH14, andHH18. A similar result was obtained for experiments investigatingrelease of the macrophage chemokine MCP3 (FIG. 16) and neutrophilschemokine Gro-α (FIG. 17), although HH1 was not active in this assay.

Based on these results new peptides were iteratively designed from thebest epptides by substitution and/or scrambling of peptide sequences.Screening of these peptides fro chemokine induction in human PBMC gavethe results presented in Table 11.

TABLE 11 Chemokine induction (pg/ml) by new peptides in human PBMC.Exoperiments were performed 2-4 times. Background values onaverage of 204 (MCP-1), 6 (MCP-3) and 196 (Gro-α) weresubtracted. Bold numbers represent significant upregulation (p < 0.05).Chemokine induction (pg/ml) by the given concentrations of peptide MCP-1MCP-3 Gro-α Name Sequence 20 μg/ml 100 μg/ml 20 μg/ml 100 μg/ml 20 μg/ml100 μg/ml Background No peptide 204 6 196 Bac2a RLARIVVIRVAR 316 442 2 29 8 HH2 VQLRIRVAVIRA 4882 10235 86 283 867 2693 1001 LVRAIQVRAVIR 5162491 0 40 88 850 1002 VQRWLIVWRIRK 2472 5566 13 141 1032 2117 1003IVWKIKRWWVGR 31 1361 0 5 65 170 1004 RFWKVRVKYIRF 300 1680 1 30 55 3361005 VQLRIRVAV 1228 4555 23 126 332 2247 1006 VQLRIWVRR 392 3004 0 58 651245 1007 WNRVKWIRR 103 247 7 28 65 170 1008 RIKWIVRFR 633 1775 1 22 109869 HH7 VRLRIRVAVRRA 894 1197 11 14 122 152 1009 AIRVVRARLVRR 634 1093 66 230 377 1010 IRWRIRVWVRRI 706 5662 3 604 149 1384 1011 RRWVVWRIVQRR579 2282 1 35 46 308 1012 IFWRRIVIVKKF 11475 30148 1103 3303 3873 75421013 VRLRIRVAV 1914 4734 22 214 609 2101 1014 RQVIVRRW 83 175 0 1 6 151015 VLIRWNGKK 113 644 0 10 42 178 1016 LRIRWIFKR 269 819 1 27 26 247HH8 VRLRIRVAVIRK 194 180 1 3 7 4 1017 KRIVRRLVARIV 585 1019 0 0 56 2501018 VRLIVAVRIWRR 8774 13041 156 604 826 2692 1019 IVVWRRQLVKNK 27 438 00 5 43 1020 VRLRIRWWVLRK 2485 2813 82 35 760 370 1021 VRLRIRVAV 158 2763 10 29 112 1022 LRIRVIVWR 52 983 0 1 10 64 1023 IRVWVLRQR 250 712 0 136 38 1024 RIRVIVLKK 285 81 1 0 20 −21 HH12 KQFRIRVRVIRK 1649 635 91 21773 172 1025 RRIVKKFQIVRR 109 284 1 3 −3 32 1026 VQWRIRVRVIKK 403 4717 1430 77 2124 1027 KKQVSRVKVW 54 1466 0 14 8 204 RK 1028 LIQRIRVRNIVK 41385 0 7 −17 34 1029 KQFRIRVRV 296 205 3 3 49 60 1030 FRIRVRVIR 139 20751 35 10 674 1031 WRWRVRVWR 875 552 9 9 172 112 1032 IRVRVIWRK 896 203 213 297 12 HH15 KRFRIRVRVIRK 61 303 0 5 17 17 1033 RRVIVKKFRIRR 1747 30161 6 359 6 1034 KQFRNRLRIVKK 434 796 0 4 60 61 1035 KRWRWIVRNIRR 15 75 01 10 27 1036 VQFRIRVIVIRK 601 968 1 39 51 137 1037 KRFRIRVRV 50 33 0 0−12 −21 1038 IVVRRVIRK 25 1552 0 41 12 518 1039 IWVIRRVWR 603 2420 13 67469 1717 1040 FQVVKIKVR 74 1143 0 9 2 264 HH18 IWVIWRR 1111 9608 32 431865 2964 1041 VIWIRWR 146 1218 7 53 110 450 1042 IVWIWRR −7 12 3 3 44 91043 WIVIWRR 98 1998 0 21 26 881 1044 RRWIVWI 1561 5024 115 261 19631545 1045 RWWRIVI −2 989 0 31 13 435 1046 WIRVIRW 46 449 1 4 38 147 1047IIRRWWV 8 130 0 0 −1 −3 1048 IRWVIRW 96 38 0 0 7 −11 HH1 QRLRIRVAVIRA 02 35 49 45 516 HH3 VRFRIRVAVIRA 0 2 26 38 19 179 HH4 VRWRIRVAVIRA 7 30157 62 333 370 HH13 HQFRFRFRVRRK 1 0 40 54 15 42 HH14 HQWRIRVAVRRH 0 39140 273 53 1279 HH17 KIWVRWK 0 0 37 36 48 132 HHC-8 KIWWWWRKR 68 835 1 49 −11 HHC-9 RWRRWKWWL 9 4493 −1 48 −25 5 HHC-10 KRWWKWIRW 48 3210 −1 49−11 2 HHC-20 WRWWKIWKR 290 974 8 12 169 33 HHC-36 KRWWKWWRR 38 168 1 119 −8 HHC-45 WKRWWKKWR −9 161 −2 −2 −15 −32 HHC-48 WKKWWKRRW 2 12 −1 −1−8 −10 HHC-53 FRRWWKWFK −26 391 −1 5 −23 2 HHC-57 LRWWWIKRI 146 1364 0 921 18 HHC-66 RKRLKWWIY 351 355 0 0 −12 −23 HHC-69 KKRWVWIRY 440 245 1 017 −3 HHC-71 KWKIFRRWW −6 99 0 2 −17 1 HHC-75 RKWIWRWFL 1313 6140 34 322554 1683 HHC-77 IWWKWRRWV 98 4548 0 23 −22 50 HHC-100 RRFKFIRWW 179 197−1 0 −18 −43 HHC-123 AVWKFVKRV 46 204 0 3 −25 17 HHC-126 AWRFKNIRK 314104 3 0 75 −2 HHC-133 KRIMKLKMR 195 444 0 2 15 81 HHC-142 AIRRWRIRK 67−16 1 0 59 22 HHC-148 VVLKIVRRF 25 210 1 3 55 60

These immunomodulatory activities led to protection against infectionsby S. aureus. Briefly a mouse model of aggressive bacterial infection,widely used to assess antibiotic efficacy, was utilized. Mice weretreated with 1.6×10¹⁰ CFU of S. aureus intraperitoneally as describedpreviously (Scott, M. G. et al., 2007. An anti-infective peptide thatselectively modulates the innate immune response. Nature Biotechnology25: 465-472). Four hours post infection they received a dose of 4 mg/kgpeptide IP. The infection was allowed to progress for 4 or 24 hoursafter which mice were euthanaised and plate counts of staphylococcisurviving in the peritoneum were determined. FIG. 18 shows results forpeptides HH2, HH18 and HH17, while FIG. 19 shows results for 1002 and1012.

Amongst the preferred twelve amino acid immunomodulatory peptides, aclear pattern of related peptides were found that obviously representedminor substitutions, deletions or additions to a base sequencerepresented by SEQ ID NO: 2. Thus these peptides have a clear unitaryrelationship. In the following sequence alignments bolded lettersrepresent amino acids that are identical or represent conservativesubstitutions (i.e., hydrophobic amino acid substitutions A, L, V, W, I,or F or charge substitutions R or K).

HH2 VQLR-IRV-AVIRA SEQ ID NO: 2 1001 LV--RAIQVRAVIR SEQ ID NO: 1213 1002VQ-RWLIV-WRIRK SEQ ID NO: 1214 1010 IRWR-IRVW-VRRI SEQ ID NO: 1222 1012IFWRRI-V-IVKKF SEQ ID NO: 1224 1018 VRLI-VAVR-IWRR SEQ ID NO: 1230 1020VRLR-IR-WWVLRK SEQ ID NO: 1232 HH12 KQFR-IRVR-VIRK SEQ ID NO: 12 1026VQWR-IRVR-VIKK SEQ ID NO: 1238

Amongst the preferred nine amino acid immunoimodulatory peptides, aclear pattern of related peptides were found that obviously representedminor substitutions, deletions or additions to a base sequencerepresented by SEQ ID NO: 1225. Thus these peptides have a clear unitaryrelationship. In the following sequence alignments bolded lettersrepresent amino acids that are identical or represent conservativesubstitutions (i.e, hydrophobic amino acid substitutions A, L, V, W, I,or F; or charge substitutions R or K).

1013 VRLRIRVAV SEQ ID NO: 1225 1005 VQLRIRVAV SEQ ID NO: 1217 1006VQLRIWVRR SEQ ID NO: 1218 1030 FRIRVRVIR SEQ ID NO: 1242 1031 WRWRVRVWRSEQ ID NO: 1243 1032 IRVRV-IWRK SEQ ID NO: 1244

Amongst the preferred seven amino acid immunoimodulatory peptides, aclear pattern of related peptides were found that obviously representedminor substitutions, deletions or additions to a base sequencerepresented by SEQ ID NO: 18. Thus these peptides have a clear unitaryrelationship. In the following sequence alignments bolded lettersrepresent amino acids that are identical or represent conservativesubstitutions (i.e., hydrophobic amino acid substitutions A, L, V, W, I,or F; or charge substitutions R or K).

HH18 IWVIWRR SEQ ID NO: 18 1041 VIWIRWR SEQ ID NO: 1253 1043 WIVIWRRSEQ ID NO: 1255

EXAMPLE 9

Adjuvanticity as a Result of Enhancement of Innate Immunity

It is well accepted that vaccine immunization is best achieved byco-administration of an adjuvant. The precise mechanism by which theseadjuvants work has eluded immunologists but appears to work in part byupregulating elements of innate immunity that smooth the transition toadaptive (antigen-specific) immunity (Bendelac A and R. Medzhitov. 2002.Adjuvants of immunity: Harnessing innate immunity to promote adaptiveimmunity J. Exp. Med. 195:F19-F23). Within this concept there areseveral possible avenues by which adjuvants might work including theattraction of immune cells into the site at which a particular antigenis injected, through e.g., upregulation of chemokines, the appropriateactivation of cells when they reach that site, which can be caused bylocal cell or tissue damage releasing endogenous adjuvants or throughspecific cell activation by the adjuvants, and the compartmentalizationof immune responses to the site of immunization (the so-called “depot”effect). Due to their ability to selectively modulate cell responses,including induction of chemokine expression, cationic host defencepeptides such as human LL-37 and defensins, have been examined foradjuvant activity and demonstrated to enhance adaptive immune responsesto a variety of antigens (Bowdish D M, D J Davidson, R E W Hancock.2006. Immunomodulatory properties of defensins and cathelicidins. CurrTop Microbiol Immunol 2006:27-66). Therefore we studies the ability ofour small host defence peptides to upregulate adjuvant responses in bothhuman PBMC and cord blood mononuclear cells (CBMC) (representing theresponses of blood cells from neonates), both alone (Table 10 and 11)and in combination with other proposed adjuvant agents that might workthrough other mechanisms such as CpG oligodeoxy ribonucleotides (TLR9agionists that activate cells through interaction with TLR9 and fit into3 different classes A=molecule 2336, B=10103 and C=2395), andpolyphosphazene P6 (which induces a depot effect). The resultsdemonstrate a variety of peptides that lead to upregulation of chemokineproduction (Table 10), and most of these are either additively enhancingchemokine induction in the presence of CpG or the combination of CpG andP6 (Table 12), or actually demonstrate significant synergy (bolded inTable 12). In particular HH2 showed excellent ability to upregulatechemokine production, significant synergy with CpG (particularly CpG-B)in PBMC and CBMC, and an ability to enhance antigen specific responsesin mose model experiments using pertussis toxin as an adjuvant.

TABLE 12 Potential adjuvant properties (ability to induce cytokines andchemokines) of peptides in combination with polyphosphazines (P6) andCpG oligonucleotides of classes A-C. Cytokines induced (pg/ml) TreatmentGro-α MCP-3 MCP-1 IL-8 IL-6 TNF-α Control PBMC Background 28 6 13 81 130 Peptide alone HH2 21 0 54 624 23 0 (20 ug/mL) HH3 8 0 8 490 7 0 HH18143 3 64 2558 180 56 HH17 0 3 91 146 0 7 CpG alone CpG-A (2336) 101 66462 41 −2 26 (5 ug/mL) CpG-B (10103) 73 32 250 447 −3 12 CpG-C (2395)123 71 350 174 −5 24 HH2 + CpG HH2 + CpG-A 152 90 570 393 16 48 HH2 +CpG-B 280 453 772 344 7 56 HH2 + CpG-C 274 352 705 267 10 73 HH3 + CpGHH3 + CpG-A 110 47 593 292 −3 25 HH3 + CpG-B 198 207 683 522 0 36 HH3 +CpG-C 181 158 587 823 1 22 HH18 + CpG HH18 + CpG-A 138 163 466 900 18 53HH18 + CpG-B 138 368 614 2850 72 35 HH18 + CpG-C 119 339 534 1391 28 46HH17 + CpG HH17 + CpG-A 76 38 315 227 −2 40 HH17 + CpG-B 95 33 285 13231 26 HH17 + CpG-C 100 93 524 678 0 22 Control PBMC Background 5 ND ND 9ND 210 Peptide alone 1012 26 ND ND 8 ND −90 (5 ug/mL) 1002 8 ND ND 10 ND282 Polyphosphazene P6 5 ug/mL 0 ND ND −1 ND 83 P6 P6 10 ug/mL 0 ND ND 1ND 35 CpG (5 ug/mL) CpG-B(10103) 205 ND ND 18 ND 968 Combination 1012 +P6 + CpG-B 240 ND ND 48 ND 1478 5 ug/mL 1002 + P6 + CpG-B 16 ND ND 23 ND971 P6 10 ug/mL, CpG 1002 + P6 + CpG-B 73 ND ND 26 ND 373 and peptide at1012 + P6 + CpG-B 77 ND ND 53 ND 1170 5 ug/mL Control CBMC Background 25110 121 413 20 8 CBMC Peptide HH2 45 −40 131 104 11 −4 alone (20 ug/mL)HH3 37 −43 249 471 14 −8 HH18 55 −24 54 372 7 −4 HH17 12 −41 25 41 −2 −8CBMC CpG-A (2336) −4 30 165 473 311 2 CpG alone CpG-B (10103) 133 99 204674 289 15 (5 ug/mL) CpG-C (2395) 96 134 202 908 259 10 CBMC HH2 + CpG-A75 164 410 406 353 12 HH2 + CpG HH2 + CpG-B 149 347 489 433 873 26 HH2 +CpG-C 52 221 504 734 722 21 CBMC HH3 + CpG-A 50 149 539 427 408 6 HH3 +CpG HH3 + CpG-B 126 182 583 704 725 14 HH3 + CpG-C 40 159 551 840 697 26CBMC HH18 + CpG-A 81 89 308 688 469 5 HH18 + CpG HH18 + CpG-B 171 146363 998 461 6 HH18 + CpG-C 122 126 371 1248 559 5 CBMC HH17 + CpG-A 17−8 393 −38 412 −1 HH17 + CpG HH17 + CpG-B 94 91 339 1804 241 3 HH17 +CpG-C 86 97 385 2012 405 5 All experiments were performed with humanPBMC except those indicated as having been done with CBMC. The indicatedbackgrounds were subtracted from the measurements with differentadjuvants alone and in combination. Bolded numbers represent apparentlysynergistic combinations. ND = Not done.

APPENDIX

Non-Natural Amino Acids

Tryptophan Variants

-   2. DL-7-azatryptophan-   3. β-(3-benzothienyl)-L-alanine-   4. β-(3-benzothienyl)-D-alanine-   5. 5-benzyloxy-DL-tryptophan-   6. 7-benzyloxy-DL-tryptophan-   7. 5-bromo-DL-tryptophan-   8. 5-fluoro-DL-tryptophan-   9. 6-fluoro-DL-tryptophan-   10. 5-hydroxy-L-tryptophan-   11. 5-hydroxy-DL-tryptophan-   12. 5-methoxy-DL-tryptophan-   13. α-methyl-DL-tryptophan-   14. 1-methyl-DL-tryptophan-   15. 5-methyl-DL-tryptophan-   16. 6-methyl-DL-tryptophan-   17. 7-methyl-DL-tryptophan-   18. D-1,2,3,4-tetrahydronorharman-3-carboxylic acid-   19. DL-6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid-   20. 5-Hydroxytryptophan: 2-Amino 3-[5-hydroxyindolyl]-propionic acid-   21. L-Neo-Tryptophan-   22. D-Neo-Tryptophan    Phenylalanine and Tyrosine Variants-   24. 4-aminomethyl-L-phenylalanine-   25. 4-aminomethyl-D-phenylalanine-   26. 4-amino-L-phenylalanine-   27. 4-amino-D-phenylalanine-   28. 3-amino-L-tyrosine-   29. 4-bromo-L-phenylalanine-   30. 4-bromo-D-phenylalanine-   31. 4-bis(2-chloroethyl)amino-L-phenylalanine-   32. 2-chloro-L-phenylalanine-   33. 2-chloro-D-phenylalanine-   34. 4-chloro-L-phenylalanine-   35. 4-chloro-D-phenylalanine-   36. 3-chloro-L-tyrosine-   37. 3,4-dichloro-L-phenylalanine-   38. 3,4-dichloro-D-phenylalanine-   39. 3,4-difluoro-L-phenylalanine-   40. 3,4-difluoro-D-phenylalanine-   41. 3,4-dihydroxy-L-phenylalanine-   42. 3,5-diiodo-L-thyronine-   43. 3,5-diiodo-D-tyrosine-   44. 3,4-dimethoxy-L-phenylalanine-   45. 3,4-dimethoxy-DL-phenylalanine-   46. O-ethyl-L-tyrosine-   47. O-ethyl-D-tyrosine-   48. 2-fluoro-L-phenylalanine-   49. 2-fluoro-D-phenylalanine-   50. 4-fluoro-L-phenylalanine-   51. 4-fluoro-D-phenylalanine-   52. 3-fluoro-DL-tyrosine-   53. L-homophenylalanine-   54. D-homophenylalanine-   55. 2-hydroxy-3-methyl-L-phenylalanine-   56. 2-hydroxy-3-methyl-D-phenylalanine-   57. 2-hydroxy-3-methyl-DL-phenylalanine-   58. 2-hydroxy-4-methyl-L-phenylalanine-   59. 2-hydroxy-4-methyl-D-phenylalanine-   60. 2-hydroxy-4-methyl-DL-phenylalanine-   61. 2-hydroxy-5-methyl-L-phenylalanine-   62. 2-hydroxy-5-methyl-D-phenylalanine-   63. 2-hydroxy-5-methyl-DL-phenylalanine-   64. β-hydroxy-DL-phenylalanine (DL-threo-3-phenylserine)-   65. 7-hydroxy-(S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid    (hydroxy-Tic-OH)-   66. 7-hydroxy-(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid    (hydroxy-D-Tic-OH)-   67. 4-iodo-L-phenylalanine-   68. 4-iodo-D-phenylalanine-   69. 3-iodo-L-tyrosine-   70. α-methyl-3-methoxy-DL-phenylalanine-   71. α-methyl-4-methoxy-L-phenylalanine-   72. α-methyl-4-methoxy-DL-phenylalanine-   73. α-methyl-L-phenylalanine-   74. α-methyl-D-phenylalanine-   75. β-methyl-DL-phenylalanine-   76. α-methyl-DL-tyrosine-   77. O-methyl-L-tyrosine-   78. O-methyl-D-tyrosine-   79. 4-nitro-L-phenylalanine-   80. 4-nitro-D-phenylalanine-   81. 3-nitro-L-tyrosine-   82. (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-Tic-OH)-   83. (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (D-Tic-OH)-   84. L-thyronine-   85. DL-thyronine-   86. L-thyroxine-   87. D-thyroxine-   88. 2,4,5-trihydroxy-DL-phenylalanine-   89. 3,5,3′-triiodo-L-thyronine-   90. DL-m-tyrosine-   91. DL-o-tyrosine-   92. 2-(trifluoromethyl)-L-phenylalanine-   93. 2-(trifluoromethyl)-D-phenylalanine-   94. 2-cyano-L-phenylalanine-   95. 2-cyano-D-phenylalanine-   96. 2-methyl-L-phenylalanine-   97. 2-methyl-D-phenylalanine-   98. 3-(trifluoromethyl)-L-phenylalanine-   99. 3-(trifluoromethyl)-D-phenylalanine-   100. 3-cyano-L-phenylalanine-   101. 3-cyano-D-phenylalanine-   102. 3-fluoro-L-phenylalanine-   103. fluoro-D-phenylalanine-   104. 3-methyl-L-phenylalanine-   105. 3-methyl-D-phenylalanine-   106. 4-benzoyl-L-phenylalanine-   107. 4-benzoyl-D-phenylalanine-   108. 4-(trifluoromethyl)-L-phenylalanine-   109. 4-(trifluoromethyl)-D-phenylalanine-   110. 4-cyano-L-phenylalanine-   111. 4-cyano-D-phenylalanine-   112. 4-methyl-L-phenylalanine-   113. 4-methyl-D-phenylalanine-   114. 2,4-dichloro-L-phenylalanine-   115. 2,4-dichloro-D-phenylalanine-   116. 3,5-diiodo-L-tyrosine OSu    Arginine and Lysine Variants-   118. L-2-amino-3-guanidinopropionic acid-   119. L-2-amino-3-ureidopropionic acid (Albizziin)-   120. L-citrulline-   121. DL-citrulline-   122. 2,6-diaminoheptanedioic acid (mixture of isomers)-   123. N-ω,ω-dimethyl-L-arginine (symmetrical)-   124. N-ε,ε-dimethyl-L-lysine hydrochloride salt-   125. α-methyl-DL-ornithine-   126. N-ω-nitro-L-arginine-   127. N-ω-nitro-D-arginine-   128. N-δ-benzyloxycarbonyl-L-ornithine-   129. (N-δ-)-L-ornithine-   130. (N-δ-)-D-ornithine-   131.    (N-δ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-ornithine    (D-Orn-(Dde)-OH)-   132. L-ornithine (Orn( )OH)-   133. (N-d-4-methyltrityl)-L-ornithine (Orn(Mtt)-OH)-   134. (N-d-4-methyltrityl)-D-ornithine (D-Orn(Mtt)-OH)    Proline Variants-   136. cis-4-amino-L-proline methyl ester hydrochloride salt-   137. trans-4-amino-L-proline methyl ester hydrochloride salt-   138. (S)-azetidine-2-carboxylic acid-   139. trans-4-cyano-L-proline-   140. cis-4-cyano-L-proline methyl ester-   141. trans-4-cyano-L-proline methyl ester-   142. 3,4-dehydro-L-proline-   143. (R)-5,5-dimethylthiazolidine-4-carboxylic acid-   144. (4S,2RS)-2-ethylthiazolidine-4-carboxylic acid-   145. trans-4-fluoro-L-proline-   146. (2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid    (trans-3-hydroxy-L-proline)-   147. (2S,4S)-(−)-4-hydroxypyrrolidine-2-carboxylic acid    (cis-4-hydroxy-L-proline)-   148. (2S,4R)-(−)-4-hydroxypyrrolidine-2-carboxylic acid    (trans-4-hydroxy-L-proline)-   149. (2R,4R)-(+)-4-hydroxypyrrolidine-2-carboxylic acid    (cis-4-hydroxy-D-proline)-   150. (2S,4R)-(−)-4-t-butoxypyrrolidine-2-carboxylic acid    (trans-4-t-butoxy-L-proline)-   151. (2S,5RS)-5-methylpyrrolidine-2-carboxylic acid-   152. (4S,2RS)-2-methylthiazolidine-4-carboxylic acid-   153. (2S,3R)-3-phenylpyrrolidine-2-carboxylic acid-   154. (4S,2RS)-2-phenylthiazolidine-4-carboxylic acid-   155. (S)-thiazolidine-2-carboxylic acid-   156. (R)-thiazolidine-2-carboxylic acid-   157. (S)-thiazolidine-4-carboxylic acid-   158. (R)-thiazolidine-4-carboxylic acid (L-thioproline)-   159. α-allyl-DL-proline-   160. α-benzyl-DL-proline-   161. α-(2-bromobenzyl)-DL-proline-   162. α-(4-bromobenzyl)-DL-proline-   163. α-(2-chlorobenzyl)-DL-proline-   164. α-(3-chlorobenzyl)-DL-proline-   165. α-(diphenylmethyl)-DL-proline-   166. α-(4-fluorobenzyl)-DL-proline-   167. α-methyl-DL-proline-   168. α-(4-methylbenzyl)-DL-proline-   169. α-(1-naphthylmethyl)-DL-proline-   170. α-propyl-DL-proline-   171. 4-benzyl-L-pyroglutamic-   172. 4-(2-bromobenzyl)-L-pyroglutamic acid benzyl ester-   173. 4-(4-bromobenzyl)-L-pyroglutamic acid benzyl ester-   174. 4-(4-methylbenzyl)-L-pyroglutamic acid benzyl ester    Miscellaneous Heterocyclic Amino Acids-   176. α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid-   177. 2-amino-α-(methoxyimino)-4-thiazoleacetic acid (predominantly    syn)-   178. 5-aminoorotic acid-   179. 2-aminopyridyl-3-carboxylic acid (2-aminonicotinic acid)-   180. 6-aminopyridyl-3-carboxylic acid (6-aminonicotinic acid)-   181. 2-aminothiazole-4-acetic acid-   182. (S)-azetidine-2-carboxylic acid-   183. azetidine-3-carboxylic acid-   184. 4-carboxymethylpiperazine-   185. 4-carboxymethylpiperazine-   186. 2-carboxypiperazine-   187. 3-carboxypiperidine-   188. indoline-2-carboxylic acid-   189. L-mimosine-   190. 4-phenylpiperidine-4-carboxylic acid-   191. (S)-(−)-piperidine-2-carboxylic acid (L-(−)-pipecolic acid)-   192. (R)-(+)-piperidine-2-carboxylic acid (D-(+)-pipecolic acid)-   193. (RS)-piperidine-2-carboxylic acid (DL-pipecolic acid)-   194. piperidine-4-carboxylic acid (isonipecotic acid)    Analogs of Alanine, Glycine, Valine, and Leucine-   196. 3-(2-furyl)-D-Ala-OH-   197. 3-cyclopentyl-DL-Ala-OH-   198. 3-(4-quinolyl)-DL-Ala-OH-   199. 3-(4-quinolyl)-DL-Ala-OH dihydrochloride dihydrate-   200. 3-(2-quinolyl)-DL-Ala-OH-   201. 3-(2-quinoxalyl)-DL-Ala-OH-   202. α-allyl-L-alanine-   203. L-allylglycine-   204. L-allylglycine dicyclohexylammonium salt-   205. D-allylglycine-   206. D-allylglycine dicyclohexylammonium salt-   207. L-α-aminobutyric acid (Abu-OH)-   208. D-α-aminobutyric acid (D-Abu-OH)-   209. DL-β-aminobutyric acid (DL-β-Abu-OH)-   210. γ-aminobutyric acid (γ-Abu-OH)-   211. α-aminoisobutyric acid (Aib-OH)-   212. DL-β-aminoisobutyric acid (DL-β-Aib-OH)-   213. Di-N-α-aminomethyl-L-alanine-   214. 2-amino-4,4,4-trifluorobutyric acid-   215. 3-amino-4,4,4-trifluorobutyric acid-   216. β-(3-benzothienyl)-L-alanine-   217. β-(3-benzothienyl)-D-alanine-   218. t-butyl-L-alanine-   219. t-butyl-D-alanine-   220. L-t-butylglycine-   221. D-t-butylglycine-   222. β-cyano-L-alanine-   223. β-cyclohexyl-L-alanine (Cha-OH)-   224. β-cyclohexyl-D-alanine (D-Cha-OH)-   225. L-cyclohexylglycine (Chg-OH)-   226. D-cyclohexylglycine (D-Chg-OH)-   227. β-cyclopentyl-DL-alanine-   228. β-cyclopenten-1-yl-DL-alanine-   229. β-cyclopropyl-L-alanine-   230. cyclopropyl-DL-phenylglycine-   231. DL-dehydroarmentomycin-   232. 4,5-dehydro-L-leucine-   233. L-α,γ-diaminobutyric acid (Dab-OH)-   234. D-α,γ-diaminobutyric acid (D-Dab-OH)-   235. Di-L-α,γ-diaminobutyric acid (Dab( )-OH)-   236. Di-D-α,γ-diaminobutyric acid (D-Dab( )-OH)-   237. (N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid (Dab(Aloc)-OH)-   238. (N-γ-)-L-α,γ-diaminobutyric acid (Dab( )-OH)-   239.    (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric    acid (Dab(Dde)-OH)-   240. (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid (Dab(Mtt)-OH)-   241. (N-γ-)-D-α,γ-diaminobutyric acid (D-Dab( )-OH)-   242.    (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric    acid (D-Dab(Dde)-OH)-   243. (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid (D-Dab(Mtt)-OH)-   244. L-α,β-diaminopropionic acid (Dap-OH)-   245. D-α,β-diaminopropionic acid (D-Dap-OH)-   246. Di-L-α,β-diaminopropionic acid (Dap( )-OH)-   247. Di-D-α,β-diaminopropionic acid (D-Dap( )-OH)-   248. (N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid    (Dap(Aloc)-OH)-   249. (N-β-)-L-α,β-diaminopropionic acid (Dap( )-OH)-   250. β-(1-naphthyl)-D-alanine (D-1-Nal-OH)-   251. β-(2-naphthyl)-L-alanine (2-Nal-OH)-   252. β-(2-naphthyl)-D-alanine (D-2-Nal-OH)-   253. L-phenylglycine (Phg-OH)-   254. D-phenylglycine (D-Phg-OH)-   255. L-propargylglycine-   256. L-propargylglycine dicyclohexylammonium salt-   257. D-propargylglycine-   258. D-propargylglycine dicyclohexylammonium salt-   259. β-(2-pyridyl)-L-alanine (L-2-pyridylalanine)-   260. β-(2-pyridyl)-D-alanine (D-2-pyridylalanine)-   261. β-(3-pyridyl)-L-alanine (L-3-pyridylalanine)-   262. β-(3-pyridyl)-D-alanine (D-3-pyridylalanine)-   263. β-(4-pyridyl)-L-alanine (L-4-pyridylalanine)-   264. β-(4-pyridyl)-D-alanine (D-4-pyridylalanine)-   265. β-(2-thienyl)-L-alanine (Thi-OH)-   266. β-(2-thienyl)-D-alanine (D-Thi-OH)-   267. L-(2-thienyl)glycine-   268. D-(2-thienyl)glycine-   269. L-(3-thienyl)glycine-   270. D-(3-thienyl)glycine-   271. 5,5,5-trifluoro-DL-leucine-   272. 4,4,4-trifluoro-DL-valine-   273. L-2-amino-3-(dimethylamino)propionic acid (aza-L-leucine)-   274. DL-2-amino-3-(dimethylamino)propionic acid (aza-DL-leucine)-   275.    (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic    acid (Dap(Dde)-OH)-   276. (N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid    (Dap(Dnp)-OH)-   277. (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid (Dap(Mtt)-OH)-   278. (N-β-)-L-α,β-diaminopropionic acid (Dap( )-OH)-   279. (N-β-)-D-α,β-diaminopropionic acid (D-Dap( )-OH)-   280.    (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic    acid (D-Dap(Dde)-OH)-   281. 2,5-dihydro-D-phenylglycine-   282. 2,4-dinitro-DL-phenylglycine-   283. 2-fluoro-DL-phenylglycine-   284. 4-fluoro-L-phenylglycine-   285. 4-fluoro-D-phenylglycine-   286. 3-fluoro-DL-valine-   287. 4-hydroxy-D-phenylglycine-   288. α-methyl-DL-leucine-   289. β-(1-naphthyl)-L-alanine (1-Nal-OH)-   290. β-(1-naphthyl)-D-alanine (D-1-Nal-OH)    Analogs of Benzoic Acid-   292. 2-amino-4-fluorobenzoic acid-   293. 2-amino-5-fluorobenzoic acid-   294. 2-amino-6-fluorobenzoic acid-   295. 2-amino-5-iodobenzoic acid-   296. 2-amino-3-methoxybenzoic acid-   297. 2-amino-5-methoxybenzoic acid-   298. 3-amino-4-methoxybenzoic acid-   299. 4-amino-3-methoxybenzoic acid-   300. 2-amino-3-methylbenzoic acid-   301. 2-amino-5-methylbenzoic acid-   302. 2-amino-6-methylbenzoic acid-   303. 3-amino-2-methylbenzoic acid-   304. 3-amino-4-methylbenzoic acid-   305. 4-amino-3-methylbenzoic acid-   306. 3-aminomethylbenzoic acid (Mamb-OH)-   307. 4-aminomethylbenzoic acid (Pamb-OH)-   308. 2-amino-3,4,5-trimethoxybenzoic acid-   309. Di-3,4-diaminobenzoic acid-   310. Di-3,5-diaminobenzoic acid-   311. 4-methylaminobenzoic acid-   312. 5-acetamido-2-aminobenzoic acid (5-acetamidoanthranilic acid)-   313. 2-aminobenzene-1,4-dicarboxylic acid-   314. 3-aminobenzene-1,2-dicarboxylic acid-   315. 2-aminobenzoic acid (2-Abz-OH)-   316. 3-aminobenzoic acid (3-Abz-OH)-   317. 4-aminobenzoic acid (4-Abz-OH)-   318. 2-(2-aminobenzoyl)benzoic acid-   319. 2-amino-5-bromobenzoic acid-   320. 2-amino-4-chlorobenzoic acid-   321. 2-amino-5-chlorobenzoic acid-   322. 2-amino-6-chlorobenzoic acid-   323. 3-amino-4-chlorobenzoic acid-   324. 4-amino-2-chlorobenzoic acid-   325. 5-amino-2-chlorobenzoic acid-   326. 2-amino-4,5-dimethoxybenzoic acid-   327. 2-amino-3,5-dimethylbenzoic acid-   328. 2-amino-4-fluorobenzoic acid    Miscellaneous Aromatic Amino Acids-   330. Di-2-amino-3-(2-aminobenzoyl)propionic acid-   331. 4-aminocinnamic acid (predominantly trans)-   332. 4-aminohippuric acid-   333. 3-amino-2-naphthoic acid-   334. 4-aminooxanilic acid-   335. (3-aminophenyl)acetic acid-   336. (4-aminophenyl)acetic acid-   337. 4-(4-aminophenyl)butanoic acid-   338. 3-amino-3-phenylpropionic acid-   339. (4-aminophenylthio)acetic acid-   340. (2R,3S)-2-amino-3-(phenylthio)butanoic acid-   341. Analogs of Cysteine and Methionine-   342. S-acetamidomethyl-L-penicillamine-   343. S-acetamidomethyl-D-penicillamine-   344. S-(2-aminoethyl)-L-cysteine-   345. S-benzyl-L-cysteine-   346. S-benzyl-D-cysteine-   347. S-benzyl-DL-homocysteine-   348. L-buthionine-   349. L-buthioninesulfoximine-   350. DL-buthioninesulfoximine-   351. S-n-butyl-L-cysteine-   352. S-t-butyl-L-cysteine-   353. S-t-butyl-D-cysteine-   354. S-carbamoyl-L-cysteine-   355. S-carboxyethyl-L-cysteine-   356. S-carboxymethyl-L-cysteine-   357. L-cysteic acid-   358. S-diphenylmethyl-L-cysteine-   359. L-ethionine (2-amino-4-(ethyl(thio)butyric acid)-   360. D-ethionine (D-2-amino-4-(ethyl(thio)butyric acid)-   361. S-ethyl-L-cysteine-   362. S-trityl-L-homocysteine-   363. Di-L-homocystine-   364. DL-methionine methylsulfonium chloride-   365. S-4-methoxybenzyl-L-penicillamine-   366. S-4-methoxybenzyl-L-penicillamine (Pen(4-MeOBzl)-OH)-   367. S-4-methylbenzyl-L-penicillamine dicyclohexylammonium salt    (Pen(4-MeBzl)-OH.DCHA)-   368. S-methyl-L-cysteine-   369. α-methyl-DL-methionine-   370. S-(2-(4-pyridyl)ethyl)-L-cysteine-   371. S-(2-(4-pyridyl)ethyl)-DL-penicillamine-   372. Di-seleno-L-cystine-   373. L-selenomethionine-   374. DL-selenomethionine-   375. S-trityl-L-penicillamine-   376. S-trityl-D-penicillamine-   377. Di-L-cystathion-   378. Di-DL-cystathionine    Analogs of Serine, Threonine, and Statine-   380. 2-amino-3-methoxypropionic acid-   381. L-α-methylserine-   382. D-α-methylserine-   383. (S)-2-amino-4-trityloxybutanoic acid (Hse(Trt)-OH)-   384. (RS)-2-amino-4-trityloxybutanoic acid (DL-Hse(Trt)-OH)-   385. (S)-2-amino-3-benzyloxypropionic acid-   386. (R)-2-amino-3-benzyloxypropionic acid-   387. (2S,3S)-2-amino-3-ethoxybutanoic acid-   388. 2-amino-3-ethoxybutanoic acid-   389. 2-amino-3-ethoxypropionic acid-   390. 4-amino-3-hydroxybutanoic acid-   391. (R)-2-amino-3-hydroxy-3-methylbutanoic acid-   392. (S)-2-amino-3-hydroxy-3-methylbutanoic acid-   393. (RS)-2-amino-3-hydroxy-3-methylbutanoic acid-   394. (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (Sta-OH)-   395. (2R,3R)-3-amino-2-hydroxy-5-methylhexanoic acid-   396. (2R,3S)-3-amino-2-hydroxy-5-methylhexanoic acid-   397. (2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid-   398. (2S,3S)-3-amino-2-hydroxy-5-methylhexanoic acid-   399. (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid-   400. (2R,3S)-2-amino-3-hydroxy-4-methylpentanoic acid-   401. (2S,3RS)-2-amino-3-hydroxy-4-methylpentanoic acid-   402. 2-amino-3-hydroxypentanoic acid-   403. (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoic acid-   404. (2R,3R)-3-amino-2-hydroxy-4-phenylbutanoic acid-   405. (2S,3S)-2-amino-3-methoxybutanoic acid-   406. 2-amino-3-methoxybutanoic acid-   407. (S)-2-amino-3-methoxypropionic acid    Miscellaneous Aliphatic Amino Acids-   409. α-amino-1-adamantanepropionic acid-   410. 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (mixture of    isomers)-   411. 3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid-   412. 3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid-   413. 3-endo-aminobicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid-   414. 1-aminocyclobutane-1-carboxylic acid-   415. 5-amino-1,3-cyclohexadiene-1-carboxylic acid-   416. 1-aminocyclohexane-1-carboxylic acid-   417. (±)-cis-2-aminocyclohexane-1-carboxylic acid-   418. (±)-trans-2-aminocyclohexane-1-carboxylic acid-   419. trans-4-aminocyclohexane-1-carboxylic acid-   420. (±)-cis-3-aminocyclohexane-1-carboxylic acid-   421. cis-4-aminocyclohexane-1-carboxylic acid-   422. (±)-cis-2-aminocyclohex-4-ene-1-carboxylic acid-   423. (±)-trans-2-aminocyclohex-4-ene-1-carboxylic acid-   424. cis-4-aminocyclohexane-1-acetic acid-   425. 1-aminocyclopentane-1-carboxylic acid-   426. (±)-cis-2-aminocyclopentane-1-carboxylic acid-   427. 1-aminocyclopropane-1-carboxylic acid-   428. 2-aminoheptanoic acid-   429. 7-aminoheptanoic acid-   430. 6-aminohexanoic acid (6-aminocaproic acid)-   431. 5-aminolevulinic acid-   432. trans-4-(aminomethyl)cyclohexane-1-carboxylic acid-   433. 2-aminooctanoic acid-   434. 8-aminooctanoic acid (8-Aminocaprylic acid)-   435. 3-(aminooxy)acetic acid-   436. 5-aminopentanoic acid-   437. 11-aminoundecanoic acid    β-Amino Acids-   439. β-alanine (β-Ala-OH)-   440. L-β-homoalanine (β-homoAla-OH)-   441.    (S)—N-ω-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-β-homoarginine    (β-homoArg(Pbf)-OH)-   442. N-ω-tosyl-L-β-homoarginine (β-homoArg(Tos)-OH)-   443. γ-trityl-L-β-homoasparagine (β-homoAsn(Trt)-OH)-   444. L-β-homoaspartic acid γ-t-butyl ester (β-homoAsp(OtBu)-OH)-   445. L-β-homoaspartic acid γ-benzyl ester (β-homoAsp(OBzl)-OH)-   446. L-β-homoglutamic acid δ-t-butyl ester (β-homoGlu(OtBu)-OH)-   447. L-β-homoglutamic acid δ-benzyl ester (β-homoGlu(OBzl)-OH)-   448. N-δ-trityl-L-β-homoglutamine (β-homoGln(Trt)-OH)-   449. O-t-butyl-L-β-homohydroxyproline (β-homoHyp(tBu)-OH)-   450. L-β-homoisoleucine (β-homoIle-OH)-   451. DL-β-leucine (DL-β-Leu-OH)-   452. L-β-homoleucine (β-homoLeu-OH)-   453. L-N-ω-β-homolysine (β-homoLys( )-OH)-   454. L-N-ω-2-benzyloxycarbonyl-β-homolysin (˜homoLys(Z)—OH)-   455. L-β-homomethionine (β-homoMet-OH)-   456. L-β-phenylalanine (β-Phe-OH)-   457. D-β-phenylalanine (D-β-Phe-OH)-   458. L-β-homophenylalanine (β-homoPhe-OH)-   459. L-β-homoproline (β-homoPro-OH)-   460. O-t-butyl-L-β-homoserine (β-homoSer(tBu)-OH)-   461. O-benzyl-L-β-homoserine (β-homoSer(Bzl)-OH)-   462. O-benzyl-L-β-homothreonine (β-homoThr(Bzl)-OH)-   463. L-β-homotryptophan (β-homoTrp-OH)-   464. O-t-butyl-L-β-homotyrosine (β-homoTyr(tBu)-OH)-   465. L-β-homovaline (β-homoVal-OH)-   466. (R)-3-amino-4-(3-benzothienyl)butyric acid-   467. (S)-3-amino-4-(3-benzothienyl)butyric acid-   468. 3-aminobicyclo[2.2.2]octane-2-carboxylic acid (mixture of    isomers)-   469. (R)-3-amino-4-(4-bromophenyl)butyric acid-   470. (S)-3-amino-4-(4-bromophenyl)butyric acid-   471. (R)-3-amino-4-(2-chlorophenyl)butyric acid-   472. (S)-3-amino-4-(2-chlorophenyl)butyric acid-   473. (R)-3-amino-4-(3-chlorophenyl)butyric acid-   474. (S)-3-amino-4-(3-chlorophenyl)butyric acid-   475. (R)-3-amino-4-(4-chlorophenyl)butyric acid-   476. (S)-3-amino-4-(4-chlorophenyl)butyric acid-   477. 3-amino-3-(4-chlorophenyl)propionic acid-   478. (R)-3-amino-4-(2-cyanophenyl)butyric acid-   479. (S)-3-amino-4-(2-cyanophenyl)butyric acid-   480. (R)-3-amino-4-(3-cyanophenyl)butyric acid-   481. (S)-3-amino-4-(3-cyanophenyl)butyric acid-   482. (R)-3-amino-4-(4-cyanophenyl)butyric acid-   483. (S)-3-amino-4-(4-cyanophenyl)butyric acid-   484. (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid-   485. (S)-3-amino-4-(2,4-dichlorophenyl)butyric acid-   486. (R)-3-amino-4-(3,4-dichlorophenyl)butyric acid-   487. (S)-3-amino-4-(3,4-dichlorophenyl)butyric acid-   488. (R)-3-amino-4-(3,4-difluorophenyl)butyric acid-   489. (S)-3-amino-4-(3,4-difluorophenyl)butyric acid-   490. (R)-3-amino-4-(2-fluorophenyl)butyric acid-   491. (S)-3-amino-4-(2-fluorophenyl)butyric acid-   492. (R)-3-amino-4-(3-fluorophenyl)butyric acid-   493. (S)-3-amino-4-(3-fluorophenyl)butyric acid-   494. (R)-3-amino-4-(4-fluorophenyl)butyric acid-   495. (S)-3-amino-4-(4-fluorophenyl)butyric acid-   496. (R)-3-amino-4-(2-furyl)butyric acid-   497. (S)-3-amino-4-(2-furyl)butyric acid-   498. (R)-3-amino-5-hexenoic acid-   499. (S)-3-amino-5-hexenoic acid-   500. (R)-3-amino-5-hexynoic acid-   501. (S)-3-amino-5-hexynoic acid-   502. (R)-3-amino-4-(4-iodophenyl)butyric acid-   503. (S)-3-amino-4-(4-iodophenyl)butyric acid-   504. (R)-3-amino-4-(2-methylphenyl)butyric acid-   505. (S)-3-amino-4-(2-methylphenyl)butyric acid-   506. (R)-3-amino-4-(3-methylphenyl)butyric acid-   507. (S)-3-amino-4-(3-methylphenyl)butyric acid-   508. (R)-3-amino-4-(4-methylphenyl)butyric acid-   509. (S)-3-amino-4-(4-methylphenyl)butyric acid-   510. (R)-3-amino-4-(1-naphthyl)butyric acid-   511. (S)-3-amino-4-(1-naphthyl)butyric acid-   512. (R)-3-amino-4-(2-naphthyl)butyric acid-   513. (S)-3-amino-4-(2-naphthyl)butyric acid-   514. (R)-3-amino-4-(4-nitrophenyl)butyric acid-   515. (S)-3-amino-4-(4-nitrophenyl)butyric acid-   516. (R)-3-amino-4-pentafluorophenylbutyric acid-   517. (S)-3-amino-4-pentafluorophenylbutyric acid-   518. (R)-3-amino-6-phenyl-5-hexenoic acid-   519. (S)-3-amino-6-phenyl-5-hexenoic acid-   520. (R)-3-amino-5-phenylpentanoic acid-   521. (S)-3-amino-5-phenylpentanoic acid-   522. (R)-3-amino-4-(3-pyridyl)butyric acid-   523. (S)-3-amino-4-(3-pyridyl)butyric acid-   524. (R)-3-amino-4-(4-pyridyl)butyric acid-   525. (S)-3-amino-4-(4-pyridyl)butyric acid-   526. (R)-3-amino-4-(2-thienyl)butyric acid-   527. (S)-3-amino-4-(2-thienyl)butyric acid-   528. (R)-3-amino-4-(3-thienyl)butyric acid-   529. (S)-3-amino-4-(3-thienyl)butyric acid-   530. 3-amino-3-(2-thienyl)propionic acid-   531. 3-amino-4,4,4-trifluorobutyric acid-   532. (R)-3-amino-4-(2-trifluoromethylphenyl)butyric acid-   533. (S)-3-amino-4-(2-trifluoromethylphenyl)butyric acid-   534. (R)-3-amino-4-(3-trifluoromethylphenyl)butyric acid-   535. (S)-3-amino-4-(3-trifluoromethylphenyl)butyric acid-   536. (R)-3-amino-4-(4-trifluoromethylphenyl)butyric acid-   537. (S)-3-amino-4-(4-trifluoromethylphenyl)butyric acid-   538. (R)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid-   539. (S)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid-   540. 1,2,5,6-tetrahydropyridine-3-carboxylic acid (guvacine)-   541. H-L-β-Homopro-OH HCl (S)-2-(2-Pyrrolidinyl) acetic acid    hydrochloride-   542. H-DL-β-Leu-OH (1)-3-Amino-4-methylpentanoic acid-   543. H-DL-β-Homoleu-OH (1)-3-Amino-5-methylcaproic acid-   544. H-DL-β-Phe-OH (1)-3-Amino-3-phenylpropionic acid-   545. L-Homophe-OEt HCl-   546. D-Homophe-OEt HCl-   547. N-Benzyl-L-Homophe-OEt HCl-   548. N-Benzyl-D-Homophe-OEt HCl-   549. (1)-3-(amino)-4-(4-biphenylyl)butyric acid-   550. (1)-3-Amino-4-(4-biphenylyl)butyric acid hydrochloride-   551. (+)-Ethyl (S)-2-amino-4-cyclohexylbutyrate hydrochloride-   552. (−)-Ethyl (R)-2-amino-4-cyclohexylbutyrate hydrochloride    N-α-Methyl Amino Acids-   554. N-α-methyl-L-alanine (MeAla-OH)-   555. N-α-methyl-D-alanine (D-MeAla-OH)-   556. N-α-methyl-L-alloisoleucine (MeAlloIle-OH)-   557. N-α-methyl-D-alloisoleucine (D-MeAlloIle-OH)-   558. N-α-methyl-N-ω-tosyl-L-arginine (MeArg(Tos)-OH)-   559.    N-α-methyl-N-ω-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-D-arginine    (D-MeArg(Pbf)-OH)-   560. N-α-methyl-N-ω-tosyl-D-arginine (D-MeArg(Tos)-OH)-   561. N-α-methyl-L-aspartic acid-   562. N-α-methyl-L-aspartic acid β-t-butyl ester (MeAsp(OtBu)-OH)-   563. N-α-methyl-D-aspartic acid-   564. N-α-methyl-D-aspartic acid β-t-butyl ester (D-MeAsp(OtBu)-OH)-   565. N-α-methyl-4-chloro-L-phenylalanine (Me(4-Cl-Phe)-OH)-   566. N-α-methyl-4-chloro-D-phenylalanine (D-Me(4-Cl-Phe)-OH)-   567. N-α-methyl-L-glutamic acid γ-t-butyl ester (MeGlu(OtBu)-OH)-   568. N-α-methyl-D-glutamic acid γ-t-butyl ester (D-MeGlu(OtBu)-OH)-   569. N-α-methylglycine (sarcosine; Sar-OH)-   570. N-α-methyl-N-im-trityl-L-histidine (MeHis(Trt)-OH)-   571. N-α-methyl-N-im-trityl-D-histidine (D-MeHis(Trt)-OH)-   572. N-α-methyl-trans-L-4-hydroxyproline-   573. N-α-methyl-L-isoleucine (MeIle-OH)-   574. N-α-methyl-L-leucine (MeLeu-OH)-   575. N-α-methyl-D-leucine (D-MeLeu-OH)-   576. N-α-methyl-N-ε-t-L-lysine (MeLys( )-OH)-   577. N-α-methyl-N-ε-2-chlorobenzyloxycarbonyl-L-lysine    (MeLys(2-Cl—Z)—OH)-   578. N-α-methyl-4-nitro-L-phenylalanine (MePhe(4-NO2)-OH)-   579. N-α-methyl-L-norleucine (MeNle-OH)-   580. N-α-methyl-L-norvaline (MeNva-OH)-   581. N-α-methyl-L-phenylalanine (MePhe-OH)-   582. N-α-methyl-D-phenylalanine (D-MePhe-OH)-   583. N-α-methyl-L-phenylglycine (MePhg-OH)-   584. N-α-methyl-L-proline-   585. N-α-methyl-O-benzyl-L-serine (MeSer(Bzl)-OH)-   586. N-α-methyl-O-benzyl-L-serine dicyclohexylammonium salt    (MeSer(Bzl)-OH.DCHA)-   587. N-α-methyl-O-t-butyl-L-serine (MeSer(tBu)-OH)-   588. N-α-methyl-O-t-butyl-L-threonine (MeThr(tBu)-OH)-   589. N-α-methyl-L-tryptophan (MeTrp-OH)-   590. N-α-methyl-DL-tryptophan (DL-MeTrp-OH)-   591. N-α-methyl-O-benzyl-L-tyrosine (MeTyr(Bzl)-OH)-   592. N-α-methyl-O-t-butyl-L-tyrosine (MeTyr(tBu)-OH)-   593. N-α-methyl-O-methyl-L-tyrosine (MeTyr(Me)-OH)-   594. N-α-methyl-O-benzyl-D-tyrosine (D-MeTyr(Bzl)-OH)-   595. N-α-methyl-L-valine (MeVal-OH)-   596. N-α-methyl-D-valine (D-MeVal-OH)    Amino Alcohols-   598. L-alaminol-   599. D-alaminol-   600. 2-aminobenzylalcohol-   601. 3-aminobenzylalcohol-   602. 4-aminobenzylalcohol-   603. (R)-(−)-2-aminobutanol-   604. (S)-(+)-2-aminobutanol-   605. 4-aminobutanol-   606. 4-amino-2-butanol-   607. 2-amino-5-chlorobenzylalcohol-   608. (±)-cis-2-aminocyclohexanol-   609. (±)-trans-2-aminocyclohexanol-   610. trans-4-aminocyclohexanol-   611. (1R,2S)-(−)-2-amino-1,2-diphenylethanol-   612. (1S,2R)-(+)-2-amino-1,2-diphenylethanol-   613. 2-(2-aminoethoxy)ethanol-   614. α-(1-aminoethyl)-4-hydroxybenzyl alcohol-   615. 2-amino-2-ethyl-1,3-propanediol-   616. 6-aminohexanol-   617. 1-amino-4-(2-hydroxyethyl)piperazine-   618. (1R,2S)-(+)-cis-1-amino-2-indanol-   619. (1S,2R)-(−)-cis-1-amino-2-indanol-   620. (1S,2R)-(+)-2-amino-3-methoxyphenylpropanol-   621. (±)-cis-2-aminomethylcycloheptanol-   622. (±)-1-aminomethylcyclohexanol-   623. (±)-cis-2-aminomethylcyclohexanol-   624. (±)-trans-2-aminomethylcyclohexanol-   625. (±)-cis-2-aminomethylcyclooctanol-   626. 6-amino-2-methyl-2-heptanol (heptaminol)-   627. α-aminomethyl-3-hydroxybenzyl alcohol (norphenylephrine)-   628. α-aminomethyl-4-hydroxybenzyl alcohol (octopamine)-   629. α-aminomethyl-4-hydroxy-3-methoxybenzyl alcohol    (normetaephrine)-   630. 2-amino-2-methyl-1,3-propanediol-   631. 2-amino-2-methylpropanol (β-aminoisobutanol)-   632. (1R,2R)-(−)-2-amino-1-(4-nitrophenyl)-1,3-propanediol-   633. (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol-   634. 5-aminopentanol-   635. 1-amino-3-phenoxy-2-propanol-   636. (R)-(−)-2-amino-1-phenylethanol-   637. (S)-(+)-2-amino-1-phenylethanol-   638. 2-(4-aminophenyl)ethanol-   639. (1R,2R)-(−)-2-amino-1-phenyl-1,3-propanediol-   640. (1S,2S)-(+)-2-amino-1-phenyl-1,3-propanediol-   641. 3-amino-3-phenylpropanol-   642. (RS)-3-amino-1,2-propanediol-   643. (S)-(+)-3-amino-1,2-propanediol-   644. (R)-(−)-1-amino-2-propanol-   645. (S)-(+)-1-amino-2-propanol-   646. 3-amino-1-propanol-   647.    N-ω-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-argininol    (Arg(Pbf)-ol)-   648. N-ω-tosyl-L-argininol-   649. N-β-trityl-L-asparaginol (Asn(Trt)-ol)-   650. L-asparaginol (Asn-ol)-   651. N-β-trityl-D-asparaginol (D-Asn(Trt)-ol)-   652. D-asparaginol (D-Asn-ol)-   653. L-aspartimol β-t-butyl ester (Asp(OtBu)-ol)-   654. D-aspartimol β-t-butyl ester (D-Asp(OtBu)-ol)-   655. DL-4-chlorophenylalaminol-   656. β-cyclohexyl-L-alaminol-   657. S-t-butyl-L-cysteinol (Cys(tBu)-ol)-   658. S-t-butyl-D-cysteinol (D-Cys(tBu)-ol)-   659. 1,1-diphenyl-L-alaminol-   660. L-glutaminol (Gln-ol)-   661. N-γ-trityl-L-glutaminol (Gln(Trt)-ol)-   662. L-glutamol γ-t-butyl ester (Glu(OtBu)-ol)-   663. L-glutamol γ-benzyl ester (Glu(OBzl)-ol)-   664. D-glutamol γ-t-butyl ester (D-Glu(OtBu)-ol)-   665. D-glutamol γ-benzyl ester (D-Glu(OtBu)-ol)-   666. ethanolamine (Gly-ol)-   667. N-im-t-L-histidinol-   668. N-im-trityl-L-histidinol-   669. N-im-benzyl-L-histidinol-   670. 1-hydroxyethylethoxypiperazine-   671. N-(2-hydroxyethyl)piperazine-   672. N-(2-hydroxyethyl)-1,3-propanediamine-   673. 3-endo-hydroxymethylbicyclo[2.2.1]hept-5-enyl-2-endo-amine-   674. (±)-cis-2-hydroxymethyl-4-cyclohexenyl-1-amine-   675. (±)-cis-2-hydroxymethyl-1-cyclohexylamine-   676. (±)-trans-2-hydroxymethyl-1-cyclohexylamine-   677. (±)-cis-2-hydroxymethyl-trans-4-phenyl-1-cyclohexylamine-   678. 3-hydroxypiperidine-   679. 4-hydroxypiperidine-   680. L-isoleucinol (Ile-ol)-   681. L-leucinol (leu-ol)-   682. D-leucinol (D-leu-ol)-   683. L-tert-leucinol ((S)-(−)-2-amino-3,3-dimethyl-1-butanol)-   684. N-ε-t-L-lysinol (Lys( )ol)-   685. N-ε-benzyloxycarbonyl-L-lysinol (Lys(Z)-ol)-   686. N-ε-2-cholorobenzyloxycarbonyl-L-lysinol (Lys(2-Cl—Z)-ol)-   687. N-ε-t-D-lysinol (D-LysQ-ol)-   688. N-ε-benzyloxycarbonyl-D-lysinol (D-Lys(Z)-ol)-   689. N-ε-2-cholorobenzyloxycarbonyl-D-lysinol (D-Lys(2-Cl—Z)-ol)-   690. L-methioninol (Met-ol)-   691. D-methioninol (D-Met-ol)-   692. (1R,2S)-(−)-norephedrine-   693. (1S,2R)-(+)-norephedrine-   694. L-norleucinol-   695. L-norvalinol-   696. L-phenylalaminol-   697. D-phenylalaminol (D-Phe-ol)-   698. L-phenylglycinol (Phg-ol)-   699. D-phenylglycinol (D-Phg-ol)-   700. 2-(2-piperidyl)ethanol-   701. 2-(4-piperidyl)ethanol-   702. 2-piperidylmethanol-   703. L-prolinol (Pro-ol)-   704. D-prolinol (D-Pro-ol)-   705. O-benzyl-L-serinol (Ser(Bzl)-ol)-   706. O-t-butyl-L-serinol (Ser(tBu)-ol)-   707. O-benzyl-D-serinol (D-Ser(Bzl)-ol)-   708. O-t-butyl-D-serinol (D-Ser(tBu)-ol)-   709. O-butyl-L-threoninol (Thr(tBu)-ol)-   710. O-t-butyl-D-threoninol (Thr(tBu)-ol)-   711. O-butyl-D-threoninol (Thr(tBu)-ol)-   712. L-tryptophanol (Trp-ol)-   713. D-tryptophanol (D-Trp-ol)-   714. O-benzyl-L-tyrosinol (Tyr(Bzl)-ol)-   715. O-t-butyl-L-tyrosinol (Tyr(tBu)-ol)-   716. O-benzyl-D-tyrosinol (D-Tyr(Bzl)-ol)-   717. L-valinol (Val-ol)-   718. D-valinol (D-Val-ol)    Others-   720. Norleucine-   721. Ethionine-   722. Ornithine-   723. Thi-OH (−)-(R)-4-thiazolidine-carboxylic acid-   724. 2-phosphonoglycine trimethyl ester-   725. iminodiacetic acid-   726. (1)-2-Aminoheptanedioic acid-   727. (1)-2-Aminopimelic acid-   728. 2-[2-(amino)ethoxy]ethoxy}acetic acid-   729. 8-(amino)-3,6-dioxaoctanoic acid-   730. 1-azetidine-3-carboxylic acid-   731. (1R,4S)-(+)-4-(amino)-2-cyclopentene-1-carboxylic acid-   732. cycloleucine-   733. homocycloleucine-   734. Freidinger's lactam-   735. 1,2,3,4-tetrahydronorharman-3-carboxylic acid-   736. 4-(aminomethyl)benzoic acid-   737. 3-(aminomethyl)benzoic acid-   738. 4-Abz-OH 4-(amino)benzoic acid-   739. 3-Abz-OH 3-(amino)benzoic acid-   740. 2-Abz-OH 2-(amino)benzoic acid-   741. 2-(amino)isobutyric acid-   742. 12-(amino)dodecanoic acid-   743. 8-(amino)caprylic acid-   744. 7-(amino)enanthic acid-   745. 6-(amino)caproic acid-   746. 5-(amino)pentanoic acid-   747. 4-(amino)butyric acid-   748. N′-diaminoacetic acid-   749. L-2,3-diaminopropionic acid-   750. N-β-L-2,3-diaminopropionic acid-   751. (R)-4-(amino)-3-(Z-amino)butyric acid-   752. (S)-4-(amino)-3-(Z-amino)butyric acid-   753. 1,6-hexanediamine HCl-   754. 1,5-pentanediamine-   755. N-β-phenylenediamine-   756. N-1,4-butanediamine-   757. N-1,3-propanediamine-   758. N-ethylenediamine-   759. N—N-methylethylenediamine-   760. 1-piperazine-   761. 1-homopiperazine

1. An isolated immunomodulatory peptide comprising an amino acidsequence of SEQ ID NO: 1230, or an amino acid sequence having 90%identity thereto.
 2. A pharmaceutical composition comprising theisolated immunomodulatory peptide of claim 1.